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The potency of human testicular stem cells

Chikhovskaya, J.V.

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Citation for published version (APA):Chikhovskaya, J. V. (2013). The potency of human testicular stem cells.

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Download date: 02 Jun 2020

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Chapter V

J.V. Chikhovskaya, S.K. van Daalen, C.M. Korver, S. Repping and A.M.M. van Pelt

Manuscript in preparation

Identification of Leydig stem cells from adult human testis


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Abstract

TheproductionoftestosteronebyLeydigcells(LCs)isrequiredforprogressionofspermatogenesisandmaintenanceofthebloodtestisbarrieraspartofaspecifictesticulartissue microenvironment. Furthermore, testosterone has an effect on many organsthroughoutthebody.AdultLCsareformedatpubertybyproliferationanddifferentiationofadultLCprogenitorsthatresidewithinthetesticularinterstitium.Inaddition,inanimalstudies,adultLCprogenitorshaveshowntobeabletoregeneratethematureLeydigcellpoolupontesticulardamage.Inhumans,justasinrodents,testis-specificundifferentiatedpericyte-/mesenchymal-like cells (MSC) that reside in the interstitialperivascularnichehaverecentlybeenproposedaspossibleprogenitorsforsteroidogenicadultLCs.However,a truehumanLCstemcellcapableof invitroproliferationhasnotyetbeen identified.Here,we determine the characteristics of several testicular somatic subpopulations inordertosearchforLCprogenitors. Ourfindingssuggestthat,justasinrodents,asubpopulationofplatelet-derivedgrowth factor receptor α positive (PDGFRa+;CD140A+) human testicular somatic cellscontainapopulationoflow-proliferatingbutcommittedLCprogenitors.Inaddition,wedemonstrate that cells positive for themesenchymalmarkerMelanoma cell adhesionmolecule (MCAM/CD146+),butnegativeforPDGFRa,possesshigh invitroproliferativeactivity andhavehigh expression levels of nestin, amarker known tobeexpressed inLC progenitors. Moreover, during induced LC differentiation the CD146+/CD34-/HLAABC+subpopulationlost itsCD146expressionandbeginexpressingPDGFRa,aswellasthe specificsteroidogenicenzyme3-β-hydroxysteroiddehydrogenase2(3β–HSD2)andluteinizing hormone receptor (LHR) typical for mature androgen-producing cells. Our results strongly suggest that human testicular CD146+ somatic cells contain LC stemcellsthatcanbeisolatedandpropagatedinvitro.TheseLCstemcellscanpotentiallybeusedforfuturecelltherapyapproachestorestoretestissteroidogenicfunctionincaseofhypogonadism.

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Introduction

Spermatogenesis is a continuous process of sperm production and is highlysensitive to factors such as temperature and local hormonal concentrations (WalkerWH,2011). The presence of androgens is known to be indispensable for progressionof germ cell differentiation and for maintenance of the blood-testis barrier, while itsimultaneouslyaffectsfunctionsofvariousotherorgans(PakarainenT,etal.,2005;GriffinDK,etal.,2010;WalkerWH,2011).AndrogensareproducedbyLeydigcells (LCs),whichresidewithinthetestisinterstitium. LCs occur in two forms which are present in different stages of development(HardyMP, et al.,1991; Benton L, et al.,1995; Lejeune H, et al.,1998; Zirkin BR,2010).FoetalLCs,locatedbetweenthetestiscordsuptillpuberty,arisefrommesenchymal-likeprogenitorswithin themesonephros (Mendis-HandagamaSM,et al.). These foetal LCsprovide the initialonsetof steroidogenesis required forprenataldevelopmentofmalegonads(Mendis-HandagamaSM,etal.,2001;GeRS,etal.,2005). AdultLCsareterminallydifferentiatedsteroidogeniccells.Beinghighlyspecialized,theyareno longerabletoproliferate.MatureadultLCsareformedandmaintainedbydifferentiation of a small population of stem/progenitor interstitial cells from pubertyonwards (HardyMP, et al.,1991;DavidoffMS, et al.,2004).Dataobtained fromanimalstudiessuggestalowphysiologicalturnoverofadultLCsduringadulthood.Ontheotherhand,completerestorationoftheadultLCpopulationandregenerationofsteroidogenicactivityhavebeendemonstratedinaratmodelafterinducedLCdepletionbyadministrationofethanedimethanesulphonate(EDS),knowntocauseapoptosisof matureadultLCs(TeerdsKJ,etal.,1999;DavidoffMS,etal.,2004). The functional properties and dynamic changes in the subpopulation of LCprogenitors during tissue regeneration are only known from rodent models, but it isgenerally suggested that this LC regeneration model also holds for the human testis(Davidoff MS, et al.,2004). Undifferentiated pericyte-like cells coexpressing pericyte/mesenchymal,neuronalandglial(astrocyte/oligodendrocyte)cellmarkers,thatresideinthetesticularinterstitiuminassociationwiththemicrovasculaturesystemareconsideredaspossibleprecursorsforsteroidogenicadultLC(DavidoffMS,etal.,2009). Recently, differentways to isolate steroidogenicprogenitors from rodent testistissuehavebeendescribedsuchasasidepopulationapproachusingHoechst33342orby selection/expansion of 3-β-hydroxysteroid dehydrogenase/Δ-5-4 isomerase2 (3β–HSD2)-negative,luteinizinghormonereceptor(LHR)-negativeandα-typeplatelet-derivedgrowthfactorreceptor(PDGFRa,CD140A)-positivecells.Cellsisolatedwitheachofthesemethodspossesstheabilitytodifferentiatetowardssteroidogeniccellsandcancolonizetestis interstitiumofhypogonadal recipients in therodent (LoKC,etal.,2004;GeRS,et


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al.,2006).ExistingdataontheisolationoftestisspecificMSCssuggestthatitistheoreticallypossibletoisolatecomparableprogenitorcellpopulationsfromhumantestis(GonzalezR,etal.,2009). Recently,ourgroupdemonstratedtheexistenceofbona fide mesenchymalstemcells (MSCs), so calledmultipotent stromal cells, in primary cell cultures derived fromhumantestistissue(MizrakSC,etal.,2010;ChikhovskayaJV,etal.,2012).Wehypothesizedthat human LC progenitors and possibly even LC stem cells reside within this MSCpopulation. In the current studywe aimed at identifying LC progenitors from humantestis.

Material and Methods

Testicular cell isolation, enrichment and culture TesticularcellssuspensionsusedforisolationofadultLCprogenitorswerederivedfrom frozen-thawed fragments of testicular tissue in accordance with our previouslydescribedprotocol(vanPeltAM,etal.,1996;Sadri-ArdekaniH,etal.,2009),usingonlytheinterstitialcellenrichedcellfractionrecoveredafter1stenzymaticdigestionstep.Tissuespecimenswereobtainedafteroralinformedconsentfromthreeindividuals(URO0059,URO0034 andURO0077) undergoingbilateral orchidectomyas part of prostate cancertreatment.AccordingtoDutchlaw,ethicscommitteeapprovalwasnotrequired,becauseanonymized tissue samples were used. None of these men had previously receivedchemotherapyorradiotherapy,andthehistologicalassessmentoftestistissuesectionsconfirmedthepresenceoffullspermatogenesisinallthreecases. Isolated cells were propagated under different culture conditions: (I) cultureconditionsreportedtobeefficientforpropagationofratstemLCswithminoradaptations(Ge RS, et al.,2006), briefly DMEM/F12 (Gibco), 2%FBS (Gibco), 1nM Dexamethasone(Sigma), 10ng/ul human platelet-derived growth factor β-homodimer/BB (PDGF BB)(Sigma),10ng/ulhumanrecombinantEGF,5ng/ulhuman,recombinantbFGF,1ng/ulhumanrecombinantLIFand1%ITS,1%penicillin/streptomycin;(II)Stemprocompletemedium(Sadri-ArdekaniH,etal.,2009)and(III)mediumroutinelyusedforpropagationofbonemarrowderivedMSCs(GonzalezR,etal.,2009).Primarytesticularcellswereculturedon plastic culture dishes. Cells were sorted at passage 3. The sorted subpopulationswereexpandedongrowthfactornon-reducedMatrigel(BD,Biosciences)coateddishesin medium referred as medium at culture condition I. After reaching confluency, thepropagated cellswere passaged using 0,05%Trypsin/EDTA (Gibco) and replated in 1:3ratio.

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Mesenchymal stem cells as reference MSCsderived frombonemarrowaspirateswerekindlyprovidedbyDr.HolgerJahr and were used as a positive control in the assessment of expression of specificmesenchymalmarkers.Thesecellswereculturedinaccordancewithapreviouslydescribedprotocol(ChikhovskayaJV,etal.,2012).

Flow Cytometry and Cell Sorting FlowcytometricalassayswereperformedonaFACSCantoIIflowcytometer(BDBiosciences)usingDiva™acquisitionandanalysissoftware.Primaryculturesoftesticularinterstitialcellsatpassage3weresortedwithBDFACSAriaflowcytometerforCD146+/CD34-/7AAD- and PDGFRa+(CD140A+)/CD34-/7AAD- subpopulations. The followingfluorochrome-conjugated antibodies were used for flow cytometrical assays: anti-CD31PE,anti-CD34PECy™7,anti-CD34FITC,anti-CD44PE,anti-CD45APC,anti-CD49fFITC,anti-CD73PE, anti-CD90APC, anti-CD105FITC, anti-CD106APC, anti-CD117PE, anti-CD140A(PDGFRa)PE, anti-CD146FITC, anti-CD200PE, anti-HLAA,B,CAPC, anti-HLADRAPC,anti-SSEA4PE(allfromBDPharmingen,SanJose,CA,USA).Anti-CD29FITCwasobtainedfrom eBioscience (San Diego, USA) and anti-CD133APC fromMiltenyi-Biotec (BergischGladbach, Germany, http://www.miltenybiotec.com). Anti-SSEA4 AlexaFluor 700 labelledantibodywaspurchasedfromBDPharmingenandInvitrogen.Matchedisotypecontrolswereappliedtodeterminebackgroundfluorescencelevels.7-AAD(BDPharmingen)wasusedtoexcludenon-viablecellsfromanalysis.Duetolowconcentrationofsomaticcellsinprimarytesticularcellsuspensions,150,000eventswereusedpersingleflowcytometricalanalysisinordertoprovidecorrectinterpretationduringthegatingprocedure.

In vitro differentiation of human adult LC progenitors Inordertoassaythedifferentiationabilityofthesortedsubpopulations,sortedcells were propagated for 1-3 passages before plating under differentiation cultureconditions.Foreachpatientenrichedsubpopulationsofprogenitorsweretestedfortheirabilitytodifferentiateat34°Cand37°Cinahumidifiedincubatorat5%CO2.DifferentiationofCD146+/HLAABC+/CD34-cellswasrepeatedintwoindependentexperimentsstartingfromcryopreservedcellsderivedfromthreedifferentpatients.DifferentiationofPDGFRa+/HLAABC+/CD34-cellswasperformedonceperpatient,duetolowrecoveryofthiscellsubpopulation. Mediumcompositionusedtoinducedifferentiationofsortedinterstitialprogenitorstowardssteroidogeniccellswasadoptedfromapreviouslypublishedprotocolwith adaptations towards human recombinant growth factors and hormones: brieflyDMEM/F12without phenol red (Gibco), 2%FBS (Gibco), 1nMDexamethasone (Sigma),10ng/ulhumanPDGFBB(Sigma),humanrecombinantinsulin-likegrowthfactor1(IGF-I)ting(R&D systems), human luteinizing hormone (LH) human pituitary (Sigma), 1nMTriiodothyronine(T3)(Sigma)andThyroxine(T4)(Sigma)10nM,1%ITS(Gibco),1%penicillin/


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streptomycin(Gibco)(GeRS,etal.,2006).At5,7and10daysafterthestartofdifferentiationculture,cellswereanalyzedforexpressionofdifferentiation-specificmarkersbyRT-PCR,flow cytometry and immunocytochemicalmethods. Sorted subpopulationsmaintainedunder propagation culture conditions were used as undifferentiated controls for thedifferentiationassays.

Gene Expression Analysis IsolationoftotalRNAwasperformedwiththeRNeasymini-kit(Qiagen,Hilden,Germany) accompanied with on-column DNAse treatment by RNAse-Free DNAse(Qiagen,).Forreversetranscription,1µgoftotalRNAwasusedasinputwiththeSuperscriptIIReversetranscriptasekit(Invitrogen).ConventionalPCRswereperformedusinggene-specificprimerpairs(SupplementarydataTableSIandSII). Incontrolreactionsreversetranscriptasewasomitted. The specificity of RT-PCRwas confirmedby sequencing thePCRproducts.QuantitativeRT-PCRwasperformedonaRocheLC480instrumentusingUniversalProbeLibraryassays (UniversalProbeLibraryAssayDesignCenterwww.roche-applied-science.com)(TableSII).Datawerenormalizedfortheexpressionofhypoxanthine phosphoribosyltransferase1(HPRT1)asareference.

Immunocyto- and histochemistry Paraffinsections(5µm)of4%formalinfixedtesticulartissueand4%formalinfixedculturedcellsofinterstitialfractionbeforeandafterdifferentiationwerestainedaccordingtoconventionalprotocolincludingpermeabilizationstepandomittingtheantigenretrievalprocedure.Sampleswereincubatedwithanti-3βHSDmousemonoclonalantibody(37-2,SC-10046SantaCruzBiotechnology)indilution1:500at4°Covernight.ImmunocomplexeswerevisualizedwithPoly-HRPantimouse/rabbit/ratreagent incombinationwithDAB-brightsubstratekit(ImmunoLogic).MouseIgGwasappliedasanegativecontrolfortheimmunostaining.

Statistical analysis StatisticalanalysisandgroupcomparisonswereperformedusingtheRelativeExpressionSoftwareTool(REST)(PfafflMW,etal.,2002).Dataisexpressedasmean+SEM.Ap-valueof<0.05wasconsideredstatisticallysignificant.

Results

Isolation of two populations of LC progenitors from adult testis To identify human LC progenitors, cell suspensions were obtained from testisbiopsiesafterthe1stenzymaticdigestionstep,sinceatthisstepthecellssuspensionsare

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enrichedforinterstitialcells(ICFs)(Fig.1a).Whiletheinterstitialcellfraction(ICF)containsanaugmentedamountofsomaticcells,HLAABC+somaticcellsstillrepresentedonlyupto3-5%ofthetotalICF(Fig.1b,c).Duetointensiveenzymaticdisruptionofthetesticulartissueitisimpossibletofullyavoidcontaminationwithgermcells. For identification of human LC progenitorswe choose, based on our previousfindings, CD146 (Melanoma cell adhesion molecule (MCAM)) as a surface marker toidentify the mesenchymal progenitors (Chikhovskaya JV, et al.,2012). In addition, weused the candidate surface marker PDGFRa based on a previously published studyreportingenrichment forPDGFRa+cellsasanapproach for selectionofLCprogenitorsinarodentmodel(GeRS,etal.,2006).InordertodistinguishthetargetmesenchymalLCprogenitorsfromothercells,co-labelingwasperformedwithanti-HLAABCandanti-CD34todiscriminatethesecellsfromgermcellsandendothelialcells,respectively.Consequentgating revealedonaverage235CD146+/HLAABC+/CD34- cellsper5×105 ICFs (Fig.1b,Suppl.Fig.1a).InordertoidentifyLCprogenitorsinanalogywiththerodentLCprogenitorpopulation,weattemptedtodetectcellswithaPDGFRa+/HLAABC+phenotypeintheICFsuspensions;noprominentsubpopulation(Fig.1c,)wasidentified,andonly20PDGFRa+/HLAABC+/CD34-cellsweredetectedper5×105ICFs(Fig.1c,Suppl.Fig.1a).Interestingly,wewerenotabletodetectcellsco-expressingPDGFRaandCD146intheICFsuspensionsinanypatient,suggestingthattwodistinctMSC/LCprogenitorcellpopulationsmayexistinthehumantestis. Takingintoconsiderationthatastandardisolationprocedurewith1cm3oftesticulartissueprovidesonaverage5×105ICFcells,onlyaverysmallamountofcellspossessingtheCD146+/HLAABC+/CD34- or PDGFRa+/HLAABC+/CD34- phenotype could be recovered.Therefore,wepropagatedtheinitialICFcellstodeterminethepropagationpotentialinvitroofbothcellpopulations,CD146+/HLAABC+/CD34-andPDGFRa+/HLAABC+/CD34-,inthreetypesofculturemediumI(LCprogenitormedium),II(StemPromedium)orIII(MSCmedium)aspreviouslydescribed. All tested conditions provided proliferation of the ICF cells. Flow cytometricalanalysisrevealedthatpropagationundercultureconditionIprovidednotonlyproliferationofICFsbutalsoanincreaseinthepercentageofCD146+/HLAABC+/CD34-cellswithintheIFCpopulationandtoalowerextentPDGFRa+/HLAABC+/CD34-cells(Fig.1d,e,Suppl.Fig.1b).PropagationunderconditionII(Stempromedium)andIII(MSCmedium)showedlesssufficientincreaseofthesetargetsubpopulations.AlsointhepropagatedICFpopulationfromconditionIatpassage3,nocellsco-expressingCD146andPDGFRacouldbedetected(Fig.1f,Supplfig.1b). ThesepropagatedICFcellsweresubjectedtocellsortingtorecoverthespecificsubpopulationsofCD146+/HLAABC+/CD34-andPDGFRa+/HLAABC+/CD34-cells(Fig.1g,h,Suppl.Fig.1c).ICFcellculturesatpassage3werecomposedofonlysomatic(HLAABC+)cellsandtesticulartissuessamplesfromdifferentpatientspossessedsubstantialdifferencesin


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thepercentageofCD146+andPDGFRa+cells.Interestingly,therewasnodirectrelationbetween thepercentageofobtainedCD146+/HLAABC+/CD34- orPDGFRa+/HLAABC+/CD34-cellsandtheageoftheman(Fig1.i),supportingthehypothesisthattheMSCstemcellcontentvariesgreatlyindifferentindividuals.

Characterization of CD146+ and PDGFRa+ progenitors The sorted CD146+/HLAABC+/CD34- and PDGFRa+/HLAABC+/CD34- subpopu-lationsderivedfromculturedICFcellsfromthreeindividualsweresubculturedfor5days(passage0)todeterminetheexpressionofLCprogenitor-specificmarkersbyquantitative

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Figure 1. Isolation of adult LC progenitors from human testicular tissue.(A)Cellsuspensionobtainedfromtestisbiopsyafter1stenzymaticdigestionstepduringtesticularcellisolation(interstitialcellfraction(ICF)).FlowcytometricalassayoftheICFsuspensionforexpressingCD146(B),PDGFRa(C)cellsinthesomaticHLAABC+/CD34-cellpopulation.SimilarFACSanalysesforculturedICFcellsundercultureconditionI forexpressionofCD146 (D),PDGFRa (E).Nodetectionofcellsco-expressingCD146andPDGFRAwere found (F).Fluorescence-activatedcellsortingforCD146+/CD34-(fraction6)andPDGFRa+/CD34-(fraction7)cellsubsetsfromtheCD34-gatedpopulation(fraction5)(G,H).TherecoveredCD146+/CD34-andPDGFRa+/CD34-sortedcellsarementionedforeachpatient(I).Scalebar100µm(A)

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CD146 PDGFRa SSEA4 CD29 CD73

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RT-PCR. PCR analysis of CD146-sorted cells revealed increased expression of CD146transcriptscomparedtotheICFcellpopulation,aswellasco-expressionofhighlevelsoftheintermediatefilamentNESTINandCALB2 (CALBINDIN 2, CLARETININ)(Fig.2a),bothknownmarkersforLeydigcellprogenitors.Concerningexpressionofmarkersindicatingandrogenproduction,thiscellpopulationexpressedneither3β–HSD2norSTEROIDOGENIC ACUTE REGULATORY PROTEIN (StAR), but low levels of other steroidogenic enzymeCHOLESTEROL SIDE-CHAIN CLEAVAGE ENZYME(CYTP450scc/CYP11A)(Fig.2a). The subpopulation of PDGFRa+ cells also did not express 3β–HSD2 and onlylow levelsof NESTIN andCALB 2 were found.However, expressionofCYTP450scc andStAR, bothmarkersindicatingsteroidogenicactivity,wasdetectedatmuchhigherlevelsinPDGFRa+cellscomparedtoCD146+cells,althoughthisdifferencewasnotsignificantdue to low sample numbers (Fig. 2a). Althoughwe never observed cells with surfacecoexpressionofCD146andPDGFRa,lowexpressionlevelsofCD146transcriptscouldbedetectedinthePDGFRa+subpopulation. Further analysis indicated that the sorted CD146+ subpopulation possessed asurfaceantigenexpressionprofileconsistentwiththeminimalcriteriaofMSCidentification(CD29+/CD73+/CD90+/CD105+/CD31-/CD34-/CD45-)(Fig.2b)(Dominici M, et al.,2006;Gonzalez R, et al.,2009;Chikhovskaya JV, et al.,2012). Unfortunately, cell numbers ofPDGFRa+cellsatthispointwereinsufficienttoperformflowcytometricalanalysis.

Figure 2. Chracterization of adult human LC progenitor subpopulations. (A)QuantitativeRT-PCRassayforexpressionof LC and MSC-specific markers by cellspresent within isolated subpopulationsof CD146+/CD34- and PDGFRa+/CD34-.(B) Immunophenotypeof sortedCD146+/CD34-cells.

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Propagation potential of CD146+ and PDGFRa+ progenitors in vitro In order to investigate the self renewal capacity of CD146+ and PDGFRa+ LCprogenitors,theICFspropagatedundercultureconditionIweresubjectedtocellsortingtorecoverthespecificsubpopulationsofCD146+andPDGFRa+cellsforsubcultureunderidentical culture conditions (Fig. 3a,b).Duringpropagation,major differencesbetweenthesetwosubpopulationsappeared.WhereasCD146+sortedcellswereabletoproliferateatleastuptopassage3,enrichedPDGFRa+cellsdidnotexpandin vitroandcouldonlybepropagateduptopassage1(Fig.3c). WhentheproliferatingCD146+cellpopulationwasanalyzedatdifferentpassagesfortheexpressionofcellsurfacemarkers,acleardecreaseinthepercentageofCD146+progenitorswasobserved.ThepropagatedCD146+populationdidnotobtain cells co-expressingPDGFRa(Fig.3d,f,supplFig.2).However,asmallsubpopulationofPDGFRa+/CD146- cells appeared in thepropagatedCD146+cellpopulationatpassage3 (Fig.3f).Furthermore, a small population of cells expressing the pluripotency-associated stem

Figure 3. Propagation of adult LC progenitor subpopulations. Subculture of CD146+/CD34- (A) andPDGFRa+/CD34- (B) cell subsets obtainedafter cell sorting. (C) Propagation resultsafter subcultureof the sortedCD146+/CD34-and PDGFRa+/CD34- subpopulations for allpatients.(D,E,F,G)Changesinsurfacemarkersexpression during in vitro propagation ofsortedCD146+/CD34-cells.CD146andSSEA4expression at passage 1 (D,F) and passage 3(E,G).Scalebarrepresents100µm(A).

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Individual Cell type Passage 1 Passage 2 Passage 3 URO0059 CD146+/CD34- 25 000 360 000 2 X106

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cellmarkerSSEA4wasdetectedintheCD146+populationatpassage1,whichprobablyremainedundetectableduring initialFACSanalysisdue to the lownumbersofCD146+cells(Fig.3e,Suppl.Fig.2).Furtherpropagationtopassage3causedsubsequentreductionin the percentage of CD146+ cells (Fig. 3f, Suppl Fig.2) and the percentage of SSEA4-expressingcellsdecreased(Fig.3g).Unfortunately,thenumberofculturedPDGFRA+cellswastoolowtoperformFACSanalyses.

Differentiation potential of CD146+ and PDGFRa+ progenitors in vitro The FACS sorted subpopulations of CD146+ and PDGFRa+ testicular cellsweresubjectedtoadifferentiationassayaccordingtotheprotocoldescribedfordifferentiationof rat LC progenitors (Ge RS, et al.,2006)with smallmodifications using two differenttemperatures (34⁰C and 37⁰C). Due to the limited number of FACS sorted CD146+/HLAABC+/CD34- and PDGFRa+/HLAABC+/CD34- cells at passage 0, we performed thedifferentiationassaywithCD146+cellsatpassage0 fromonepatient (URO00059) (i.e.directly recovered fromtheFACSprocedure)and for the twootherpatients,URO0034and URO0077, with subcultured CD146+ sorted cells at passage 1 and 2 respectively(correspondingto2and3weekspropagationin vitro).AdifferentiationassaywithPDGFRa+sortedcellswasperformedwithcellsatpassage1forallthreepatients.Throughouttheperiodofdifferentiation,proliferationofplatedCD146+cellswasobservedatboth34⁰Cand37⁰C,resultinginformationofsubconfluentmonolayers(Fig.4a).IncaseofPDGFRa+cellsnoproliferationunderdifferentiationcultureconditionswasobserved(Fig.4b). When CD146+ cells at passage 0 or 1 were subjected to differentiation for 5and 7 days (under 37⁰C and 34⁰C respectively) expression of 3β–HSD2 was detectedaccompanied by expression of other markers related to cell steroidogenic functionsuch asCYTP450scc, and specific LCmarkers LUTEINIZING HORMONE RECEPTOR (LHR)and INSULIN-LIKE FACTOR 3 (INSL3), also knownasRELAXIN-LIKE FACTOR (RLF) a genepreviously described as highly specificmarker ofmature LC (Ivell R, et al.,1997) (Fig.4c,e).FlowcytometricalanalysisofthesamesamplesrevealeddisappearanceofCD146+surfaceexpressionaccompaniedwiththeappearanceofPDGFRa+subpopulationvaryingbetween2-5%ofthetotalcellpool.The 3β–HSD2expressionwasdetectedonlyinsamplescontainingPDGFRa+cells(Fig.4c,f,g). Immunocytochemicalassayshowedthepresenceofsingle3β-HSD expressingcells(Fig.4h,i).Interestingly,irrespectiveofthetemperatureanddurationof thedifferentiationassay (5or7days)3β–HSD2expression levelsweresignificantlyincreased(Fig.4c). Generallyafter10-12days,thedifferentiationassayshadtobeterminatedduetomassivecellproliferation.However,overalllevelsof3β-HSD2expressionreducedaroundthistimepointat37⁰Caswellasat34⁰C,probablydue toprogressiveproliferationofcontaminating somatic cells overgrowing the differentiating progenitors. In parallel, areductionofPDGFRa-expressing cells to0,1-0,5%wasobserved compared to the total


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cell pool in this prolonged differentiation culture conditions. We observed that theinitiationofdifferentiationwashighlydependedonthecelldensitypriortoapplicationofdifferentiationmedia;inwellswithlowconfluence(<30%)differentiationdidnottakeplace, while at confluence between 50-60% differentiationwas observed. In addition,startingwithahigherpassagenumber(p=2)ofthepropagatedCD146+subpopulationalsoreducedthedifferentiationcapability.Ofnote,inoneofthethreepatients(URO0077)wewereunabletogeneratedifferentiatingcells,mostlikelybecausethissamplehadaverylowrecoveryofCD146+/HLAABC+/CD34-(Fig.3c)andthereforealowdensityatthestartofthedifferentiationassaywithcellsatp=2. When using PDGFRa+ cells in the differentiation assay, quantitative RT-PCRanalysisrevealeddetectableincreasein3β-HSD2expressioninculturesatalltestedtimepointscomparedtotheinitialpopulationatday0ofthedifferentiationassay.However,during prolonged culture under control (non-differentiation inducing) conditions, the

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E

GF

Figure 4. Differentiation of adult LC progenitor subpopulations. CD146+/CD34- (A) and PDGFRa+/CD34-(B) cells subjected into LC differentiationmedium (Day 7 of differentiation assay).Quantitative RT-PCR for expression ofdifferentiation-related markers in theCD146+/CD34- (C) and PDGFRa+/CD34-(D) sorted subpopulations after 7 daysof differentiation towards LC. (E) RT-PCRassay for expression of genes involvedin steroidogenesis. Changes in CD146and PDGFRa surface expression duringdifferentiation of CD146+/CD34- cells(F) and CD146+/CD34- cells in controlmedium (G). 3β–HSD2 expression afterdifferentiation of CD146+/CD34- cells(H) and CD146+/CD34- cells in controlmediumasnegativecontrol(I).3β–HSD2expressionafterdifferentiationofPDGFRa+/CD34-cells(K)andinPDGFRa+/CD34- cells under propagation conditionsasnegativecontrol(J).

H I

J K

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same levels of 3β-HSD2 expression could be detected, as well as expression of STAR, CYTP450scc andevenLHR atsomesamples,indicatingnodifferencesintranscriptlevelsofPDGFRa+cellsduringapplicationofdifferentiationandcontrolconditions.(Fig.4d,e).Immunocytochemical assay confirmed thepresenceof3β-HSD2 expressing cellsunderdifferentiationcultureconditionaswellasincontrolcondition(Fig.4j,k).

Discussion InadditiontotheidentificationofPDGFRa+Leydigcellprogenitors,wedescribedforthefirsttimetheidentificationandisolationofasecondtypeofadultLCprogenitorsubpopulation(CD146+/PDGFRa-cells)fromhumantesticulartissue.TheseCD146+adultLCprogenitorsarecapableofselfrenewalanddifferentiationtowardssteroidogeniccellsin vitro.TogetherthesedatasuggestthatCD146+cellsisolatedfromadulthumantestisinterstitiumcontainLCstemcells. Our results support the previously suggested hypothesis that mesenchymal,pericyte-likecellsharbouranuncommittedprogenitoroftheLClineageinadulthumantestis (DavidoffMS, et al.,2009).Our isolatedCD146+ LCprogenitors possess CD146+expression levels comparable to expression levels ofbona fide bonemarrow- derivedMSCsandcoexpresssurfacemarkersspecificforMSCs(CD105,CD73,CD90). Furthermore,sortedCD146+/HLAABC+/CD34-cellspossessexpressionofNESTINintermediatefilamentprotein,previouslyreportedasamarkerofhumanLCprogenitors,andCALB2specificfortheLClineage(DavidoffMS,etal.,1993;LoboMV,etal.,2004).ThefactthatthepopulationofCD146+cellsderivedfromtesticulartissueadditionallyshowedexpression of thesemarkers further enables enrichment of testicular cell suspensionsforLCstemcellsusingFACS.TheresemblanceofadultLCprogenitorstotesticularMSCsis also supportedby the clinicalobservationof adiposedifferentiationand/orareasofossificationwithinLCtumorscausedbytransformationofneoplasticLCs(UlbrightTM,etal.,2002).ThisphenomenonindicatesthatneoplasticLCswithintesticulartumorspossesstheabilitytotransdifferentiatetowardsmesodermallineages.Infact,thisabilityisoneofthedefininguniversalcharacteristicofMSCsderivedfromvariousorgans.Inaddition,theadiposecellsfoundintheseneoplasticLCsareusuallypositiveforsomeLCmarkerssuchasINHIBIN-A,CALB2and/orMELAN-A(DavidoffMS,etal.,1993;UlbrightTM,etal.,2002).ThesedifferentiationabilitiesandtheexpressionofspecificmarkersbothsuggestacloserelationbetweenMSCsandLCprogenitorswithintestistissue. In contrast to animal studies, our data suggest that PDGFRa+/HLAABC+/CD34-testicularsomaticcellsfromhumandonotrepresentanearlyuncommittedprogenitor,butratheracommittedcellpopulationalreadyprogressedtowardsmaturesteroidogenicLCorperhapsamixedpopulationwithmostlikelydifferentiatedLCs.Thishypothesis issupported by the detection of early LC specificmarkers (CYTP450scc, StAR) in freshly


117

sortedPDGFRacellsandspontaneousupregulationof3β–HSD2expressioninthesecellsduring thedifferentiationorduringprolongedculture.The fact that cultureconditionsthat normally trigger differentiation did not further affect the appearance of alreadydifferentiated cells suggests that this sortedpopulation is already committed andwellprogressed in differentiation towards steroidogenic LCs. Moreover, PDGFRa-sortedcellshad lowerproliferationactivitycomparedto theuncommittedCD146+progenitorsubpopulation. This observation is in agreement with a previous report describingupregulation of PDGFRa surface expression by human embryonic stem cells duringdifferentiationtowardssteroidogeniccellsin vitro(SonoyamaT,etal.,2012).Indeed,thesmallincreaseinthePDGFRapopulationduringICFpropagationmightnotbetheresultofproliferation,butofspontaneousdifferentiation.ThisisalsoinagreementwithourfindingthatduringproliferationofsubculturedCD146+cellssomeCD146-/PDGFRa+cellsalreadyspontaneouslyappearatpassage3.FurtherstudiesarerequiredtoimprovethecultureconditionsrequiredtopropagateCD146+cellswithoutinitiatingdifferentiation. Basedonour resultswehypothesize thatCD146+/HLAABC+/CD34- cells in thehumantestisrepresentapopulationofuncommittedLeydigstemcells,whilePDGFRa+/HLAABC+/CD34- cells resemble the early committed progenitor cells that derive fromtheseuncommittedCD146+/HLAABC+/CD34-cells. Indeed,theuncommittedstemcellsareabletoselfrenewandupondifferentiationtowardmatureLCthesecells losetheirCD146expressionanddisplayPDGFRaexpression(aswellasLHR)ontheircellsurfaceandupregulatetheexpressionofsteroidogenicenzymesCYTP450scc. These subsequent transitions in cell surface marker expression profiles andchanges in steroidogenic enzymesexpression in vitro, allowus to suggest amodel foradulthumanLCregeneration.WeproposethatadulthumanLCstemcellsarerepresentedbyCD146+/HLAABC+/CD34-MSCsthatpossesshighNESTINlevels.Thispoolofcells isthen thecell source formatureLC turnover inphysiologicalconditionsaswellas theirregenerationincaseofacuteLCinjury.TheirtransitiontowardsmatureLCsactsviaseveralintermediatestages/committedLCprogenitorsthatshowdownregulationofCD146andsubsequentincreasinglevelsofPDGFRA,LHand3β-HSD2(Fig.5) Inoneoutofthreeindividualswewereunabletoderive3βHSDexpressingcellsinvitro,most likelybecausethissampleprovided insufficientnumbersofCD146+cellsandsubsequentlyculturesstartedwithvery lowdensity,precludingefficientexpansiondifferentiation in vitro. This ismost likely due to variability in the retrievable amountofLCprogenitorsbetweenmen.FollowupstudiesarerequiredtoverifythequantitiesanddistributiontheseLCstemcellsin vivoinlargergroupsofmen.TheheredescribedCD146+LCprogenitorpopulationshowsstemcellabilitybydisplayingbothselfrenewalanddifferentiation towards steroidogenic cells in vitro. Future studies shouldestablishtheabilityofthisCD146+cellpopulationtoengraftrecipienttestisandrestoreandrogenproduction in vivo forinstanceusingtheluteinizinghormonereceptorknockoutmouse

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model(LuRKO)(LoKC,etal.,2004;ZhangFP,etal.,2004). TheabilitytooccupytheemptyLeydigstemcellnichesafterxenotransplantationintotheinterstitiumoftheLuRKOmousetestiswouldprovideessentialconfirmationonstemcellpropertiesoftheCD146+Leydigstemcellsubpopulation.However,thepossibilitytoachievesteroidogenesisinvivoincaseofahumantomousexenotransplantationmodelmight be challenging because of the phylogenic distance between these species andfurtherevaluationofstemcellpropertiesmayrequireallogenictransplantationstudiesusingamonkeymodel. Our findings potentially have high clinical relevance for men undergoinggonadotoxictreatment.Hypogonadismisafrequentlyobservedsideeffectofgonadotoxictreatment. Theoretically, isolation of CD146+ Leydig stem cells before gonadotoxictreatmentwouldbeanappropriatewaytopreservetheLCpoolofpatientsundergoinggonadotoxic treatment (Chatterjee R, et al.,2001; Kyriacou C, et al.,2003). Isolation, in vitropropagationandtransplantationofenrichedautologousLeydigstemcells(withorwithoutadifferentiationstepinvitro)couldthenbeusedtorestoretestissteroidogenicfunction,preventingthenecessityof life-longandrogenreplacementtherapy.Similarly,

Nestin+

Uncommitted progenitor

3 β-HSD2

Committed progenitor

Immature LC Mature LC

CD146+

PDGFRα Nestin

CD146+ CD34 -

LHR

Figure 5. Model of LC regeneration in adult human testicular tissue.Adult human LC stem cell are representedby CD146+/HLAABC+/CD34-MSCs and possess highNESTIN levels. ThesecellsarethepoolofadultstemcellsandthesourceofmatureLCturnover inphysiologicalconditionsaswellastheirregeneration in case of acute LC injury. Their transition towardsmature LC is going via several intermediate stages/committedLCprogenitorsthatshowdownregulationofCD146andincreasinglevelsofPDGFRa,LHand3β-HSD.


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autologoustransferofinvitropropagatedspermatogonialstemcells(SSCs)couldrestorespermatogenesis. Together such dual stem cell therapy would potentially overcomeinfertility in addition to the consequences of endocrine hypogonadism such as insulinresistanceandsexualdysfunction. In the present studywe identified for the first timeuncommitted Leydig stemcellfromadulthumantestis.UsingtheMSCmarkerCD146,wewereabletodistinguishtheuncommittedCD146+LCstemcellfromthecommittedPDGFRa+LCprogenitor.Theuncommitted CD146+ LC stem cell might be very important in regenerativemedicineand further basic studies on the turnover of LCs and regulationof their function.Our findings suggest that isolation of CD146+/CD34 somatic cells from primary testicularcell suspensionsobtainedafterenzymaticdigestionof testiculartissue fragments isanappropriatewaytoderivepatient-specificLCstemcells.

Acknowledgements

We thank B. Hooibrink for help in fluorescent activated cell sorting (Dept.Cell Biology and Histology Amsterdam Academical Medical Center, Amsterdam, TheNetherlands).

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12. GriffinDK,EllisPJ,DunmoreB,BauerJ,AbelMH,AffaraNA.Transcriptionalprofilingof luteinizing hormone receptor-deficient mice before and after testosterone


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APC (HLA ABC)

FITC

(CD

146)

APC (HLA ABC)

FITC

(CD

146)

PE (P

DG

FRa)

FITC

(CD

146)

PE (P

DG

FRa)

PE (P

DG

FRa)

PE-Cy7(CD34)

PE (P

DG

FRa)

FITC (CD146)

APC (HLA ABC) APC (HLA ABC) PE (PDGFRa)

PE (P

DG

FRa)

102

102

102

102

102

FITC

(CD

146)

FITC

(CD

146)

FITC

(CD

146)

FITC

(CD

146)

PE (PDGFRa)

PE (PDGFRa) SSEA4 (Alexa 700)

SSEA4 (Alexa 700)

210

10

10

2

2

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Supplementary data

Figure S1. Isotype negative controls for flow cytometrical assays(A)Primarytesticularcellsuspensionand(B)Primarytesticularcellcultureatpassage3(C)IsotypenegativecontrolsforFACSofspecificcellpopulationsfromprimarytesticularcellculture.

Figure S2. Isotype negative controls for flow cytometrical assays CD146+/CD34-enrichedsubpopulationatpassage1(A)andpassage3(B).

A

B

C

A

B

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Supplementary Table I. Primer/probes sequences used for quantitative RT-PCR

Gene IDtranscript PrimersequenceFw

PrimersequenceRv

Probe/t

Cycles Ampli-con(bp)

CD146 NM_006500.2 gtctgcgccttcttgctc ccacctccaccagctcag Probe#56;59°C 50 97

Nestin NM_006617.1 gctcaggtcctggaaggtc aagctgagggaagtcttgga Probe#30;59°C 50 143

Calbindin2 NM_001740 atcctgccaaccgaagaga gtgtcgtacttccgccaag Probe#27;59°C 50 92

3β–HSD2HSD3B2 (typeII)

NM_000198.2 gagggcttctgggtcagag tggtcctgttctggagcttag Probe#31;59°C 50 129

CytP450 NM_000781.2 gatgacctgttccgctttg cctcggggttcactacttcc Probe#89;59°C 50 91

STAR NM_000349.2 tggaagaaggagagtcagcag agctcttcatagagcctctcca Probe#19;59°C 50 125

LHR NM_000233.3 tcttccccgattaaaatacttgag aagttatcacaaatttccagaatgaa Probe#82;59°C 50 114

RLF(INSL3) NM_005543.2 cccagagatgcgtgagaagt ccagccactgtagcaactca Probe#49;59°C 50 134

HPRT1 NM_000194.2 gaccagtcaacaggggacat gtgtcaattatatcttccacaatcaag Probe#22;59°C 50 95


125

Supplementary Table II. Primer sequences used for RT-PCR

Gene IDtranscript PrimersequenceFw

PrimersequenceRv

t/pcr type

Cycles Amplicon(bp)

3β–HSD2HSD3B2(typeII) NM_000198.2 cttcctccagggatgaggcagt tggtcctgttctggagcttagaaa 58°C 35 316

CytP450 NM_000781.2 tgcagcaggaggaaggacgtg ccgagcttctccctgtaaatcgg 57°C,hotstart

35 432

STAR NM_000349.2 cgggactcagaggcgaagct gcctgttgcctcagcccctt 64°C,hotstart

35 207

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