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Optimization of umbilical cord processing techniques
and multiparametric immunophenotyping of umbilical
cord Wharton’s jelly mesenchymal stem cells for cell
therapy applications
Olivier Degoul
To cite this version:
Olivier Degoul. Optimization of umbilical cord processing techniques and multiparametric im-munophenotyping of umbilical cord Wharton’s jelly mesenchymal stem cells for cell therapy applica-tions. Cellular Biology. Université Claude Bernard - Lyon I, 2014. English. �NNT : 2014LYO10174�. �tel-02640907�
Thèsededoctoratdel'universitéClaudeBernardLyon1
EcoleDoctoraleBiologieMoléculaireIntégrativeetCellulaire
Présentéepar
OlivierDEGOUL
Pourobtenirlediplômede
Docteurdel'UniversitéLyon1
Optimisationdestechniquesdetraitementdeséchantillons
decordonombilicaletimmunophénotypage
multiparamétriquedescellulessouchesmésenchymateuses
delageléedeWhartonducordonombilicalpourdes
applicationsenthérapiecellulaire.
Sousladirectiondu
ProfesseurColinMcGuckin
etdu
DocteurNicolasForraz
Dansleslaboratoiresdu
CTIͲLyon
InstitutderechercheenThérapieCellulaire
MEYZIEUͲLYON
Soutenuele30septembre2014devantlejurycomposéde: ProfesseurChristianVacherRapporteur DocteurFlorelleGindrauxRapporteur ProfesseurMalleinͲGerinExaminateur DocteurAnneMeyExaminateur Numérod'ordre:174Ͳ2014
Optimizationofumbilicalcordprocessingtechniquesand
multiparametricimmunophenotypingofumbilicalcord
Wharton'sjellymesenchymalstemcellsforcelltherapy
applications.
UNIVERSITECLAUDEBERNARDͲ LYON1
Présidentdel’Université ViceͲprésidentduConseild’Administration ViceͲprésidentduConseildesEtudesetdelaVieUniversitaire ViceͲprésidentduConseilScientifique DirecteurGénéraldesServices M.FrançoisͲNoëlGILLY M.leProfesseurHamdaBENHADID M.leProfesseurPhilippeLALLE M.leProfesseurGermainGILLET M.AlainHELLEUCOMPOSANTESSANTE
FacultédeMédecineLyonEst–ClaudeBernard FacultédeMédecineetdeMaïeutiqueLyonSud–CharlesMérieux Facultéd’Odontologie InstitutdesSciencesPharmaceutiquesetBiologiques InstitutdesSciencesetTechniquesdelaRéadaptation DépartementdeformationetCentredeRechercheenBiologie Humaine Directeur:M.leProfesseurJ.ETIENNE Directeur:MmelaProfesseureC.BURILLON Directeur:M.leProfesseurD.BOURGEOIS Directeur:MmelaProfesseureC. VINCIGUERRA Directeur:M.leProfesseurY.MATILLON Directeur:Mme.laProfesseureAͲM.SCHOTTCOMPOSANTESETDEPARTEMENTSDESCIENCESETTECHNOLOGIE
FacultédesSciencesetTechnologies DépartementBiologie DépartementChimieBiochimie DépartementGEP DépartementInformatique DépartementMathématiques DépartementMécanique DépartementPhysique UFRSciencesetTechniquesdesActivitésPhysiquesetSportives ObservatoiredesSciencesdel’UniversdeLyon PolytechLyon EcoleSupérieuredeChimiePhysiqueElectronique InstitutUniversitairedeTechnologiedeLyon1 EcoleSupérieureduProfessoratetdel’Education InstitutdeScienceFinancièreetd'Assurances Directeur:M.F.DEMARCHI Directeur:M.leProfesseurF.FLEURY Directeur:MmeCarolineFELIX Directeur:M.HassanHAMMOURI Directeur:M.leProfesseurS.AKKOUCHE Directeur:M.GeorgesTOMANOV Directeur:M.leProfesseurH.BENHADID Directeur:M.JeanͲClaudePLENET Directeur:M.Y.VANPOULLE Directeur:M.B.GUIDERDONI Directeur:M.P.FOURNIER Directeur:M.G.PIGNAULT Directeur:M.C.VITON Directeur:M.A.MOUGNIOTTE Directeur:M.N.LEBOISNE“Whatevercourseyoudecideupon,thereisalwayssomeonetotellyouthatyouarewrong.Thereare alwaysdifficultiesarisingwhichtemptyoutobelievethatyourcriticsareright.Tomapoutacourseof actionandfollowittoanendrequirescourage.” “Donotgowherethepathmaylead;goinsteadwherethereisnopathandleaveatrail” RalphWaldoEmersonquotes(AmericanPoet,LecturerandEssayist,1803Ͳ1882) “Scientistsinvestigatethatwhichalreadyis,Engineerscreatethatwhichhasneverbeen.” AlbertEinstein(NobelPrizeforPhysicsin1921,1879Ͳ1955) “Limitationsliveonlyinourminds.Butifweuseourimaginations,ourpossibilitiesbecomelimitless.” JamiePaolinetti(AmericanSchroederIronProCycling) “Striveforperfectionineverythingyoudo.Takethebestthatexistsandmakeitbetter.Whenitdoes notexist,designit.” SirFrederickHenryRoyce(BritishEngineer,1863Ͳ1933) “Havethecouragetosayno.Havethecouragetofacethetruth.Dotherightthingbecauseitisright. Thesearethemagickeystolivingyourlifewithintegrity.” W.ClementStone(AmericanbestsellingAuthorandFounderofCombinedInsurance,1902Ͳ2002) “EngineeringproblemsareunderͲdefined,therearemanysolutions,good,badandindifferent.Theart istoarriveatagoodsolution.Thisisacreativeactivity,involvingimagination,intuitionanddeliberate choice.” SirOveNyquistArup(AngloͲDanishEngineer,1895Ͳ1988) “Thenyouwillknowthetruth,andthetruthwillsetyoufree.”John8:31Ͳ32.
Acknowledgement
Thisworkwouldcertainlynothavebeenpossibleandachievedwithoutthehelpandsupportprovided bymany,manypeoples.
I would like to first express my deepest thanks and gratitude to my supervisors Professor Colin McGuckinandDrNicoForraz.Thisworkwouldnothavebeenpossiblewithoutyou,notonlyforyour help,guidance,supportandencouragementsallalongthisprojects,evenduringtoughtimes,youwere alwaystherewhenIneededtime,helporadvicesandthosewerealwaysright,butalsobecause,since wemeetsome8yearsago,yourtrusthasbeeninspiringandthejourneyincredible.Thankyou.
I'd also like to express my thanks to Professor Vacher and Docteur Gindraux for reviewing this manuscriptandtoProfesseurMalleinͲGerinandDocteurMeyfortheirparticipationtothejury.
I would also like to thanks the people that supported me, before and during this work, starting with GianluigiAtzeni,forhisfriendshipandforsupportingme(bothEnglishandFrenchmeaning)everyday tothisdayinourlab.
I would also like to thank Professor MalleinͲGerin and his team for the fruitful collaboration initiated betweenourresearchgroups.
Within his team I would like to particularly thanks Hugo Fabre, PhD student, not only for the irreplaceablehelpheprovidedduringflowcytometryexperimentsandforthewaytheyevolvedthanks tohisskillsbutalsoforhisfriendship,supportandforouralwaysinterestingandscientificallyenriching discussionduringourlongdaysandnightinfrontofthecytometer.
I'dalsoliketothankDrAndreasMuellerforhisfriendshipinsideandoutsidethelaboratoryandforthe pleasureIhadworkingwithyouduringthetimeyouspentatCTI.
I am very grateful to the team at the Novotec laboratory, particularly to Professeur Daniel Hartmann andDrAuréliePagnonͲMinotforthework,analysisandexpertisesheprovidedonbonedifferentiated cellsandonhistologicalanalysisoftheumbilicalcordstructure. AndsincethisprojectreallystartedfarfromLyonI'dliketothanksChristinaBasford,SabaHabibollah, andMarkoStrbadfortheirfriendshipandforshowingmetheway. I'dalsoliketothanksthepeoplethatIhadthepleasuretoworkwithwhentheycameforinternshipin ourlaboratoryduringmyPhDandparticularlyVéroniqueMenet,AlicePesquet,KevinLivet,andCharles Moulinier,thankyouforthefunwehadworkingtogether.
Je souhaite également remercier l'ensemble du personnel de la maternité Natecia à Lyon, et très particulièrementMmeNathalieCorgnet,cadresageͲfemme,l'ensembledel'équipedessageͲfemmede lamaternité,etlesfamillesquigrâceàleursdonsontpermislaréalisationdeceprojetderechercheet sansquiilauraitsimplementétéimpossible.Lescollectessontparfoisunpeutropmatinalesamongout maisnoussommessibienaccueilliquecesmomentsrestentencoreàcejourunplaisir,etc'esttoujours unémerveillementrenouveléqued'assisteralavenueaumondedecesbébés.Merciàvous.
Je remercie également mes amis pour leur soutien durant ces années de thèse et particulièrement ClémentetFanny,CarolineetGaël,AurélieetFlorian,LaurianneetMarco,MikaetLucie.
Jesouhaiteégalementprofiterdecetteoccasionpourexprimermagratitudeetmonamourpourmes parents et ma famille, Papa, Maman, Stéphanie, JeanͲFrançois et Magalie, sans vous, votre affection, votre soutient, votre confiance dans mes choix, votre patience, et la motivation que vous m'avez apporté,cetravailn'auraitpasétépossible,etsanslacuriosité,lescompétences,etl'envied'allerplus loinquejetiensdevous,iln'auraitjamaisdébuté.Mercid'avoircruenmoi.
Jevousaime.
Enfin,jesouhaiteremercier,poursacompréhension,sapatience,saconfianceetsonamourcellequi pendant deux ans m'a apporté un soutient quotidien et a sus me motiver pendant les moments difficiles.Alice,Jet'aime.
Abstract
With around 136,000,000 births per year worldwide stem cells isolated from neonatal tissues such as theumbilicalcordhavemanyadvantagesintermofaccessandavailability.
The umbilical cord contains a connective tissue present in large quantities surrounding the blood vessels,theWharton’sjelly,demonstratedasaveryrichsourceofMesenchymalStemCells(MSCs).
Those multipotent stem cells isolated from the umbilical cord have shown selfͲrenewal and differentiationcapabilityintoarangeofcellsinherentpotentialforcelltherapyapplicationsnotonlyas differentiated cells but also thanks to potent capabilities for immunomodulation and regulation of inflammation.
In this work we designed process and tools allowing efficient use of umbilical cord samples and the isolationofstandardizedWharton'sJellytissueexplants.
WestudiedtechniquesimprovingMSCisolationfromtissueexplantsanddevelopedaninͲvitroculture protocol,freeofFoetalBovineSerum(FBS)andotheranimalsourcedproductsandthusmorelikelyto beusedinclinicalsettings.
We developed a multiparametric flow cytometry protocol dedicated to phenotyping the surface expressionof27differentantigenspresentonWharton'sJellyMSCsandtostudytheeffectofseveral inͲvitrocultureconditionsandeventsontheirsurfacephenotype. ThemultiparametricprotocolwasusedtocomparesurfaceexpressionofMSCsisolatedfromWharton's jelly,bonemarrowandskinharvestedfibroblasts. Finally,clinicaldevelopmentofhumanWJͲMSCinabonetissuedifferentiationprotocolwasevaluated forcleftpalaterepair,demonstratingtheusefulapplicationofthistissuesource.
Résumé:
Avecenviron136,000,000denaissancesannuellesdanslemonde,lescellulessouchesisoléesàpartir des tissus néonataux tels que le cordon ombilical présentent de nombreux avantages en terme d'accessibilitéetdedisponibilité.
Lecordonombilicalcontientuntissusdesoutientprésentengrandequantitéetentourantlesvaisseaux sanguin,lageléedeWharton,quiaétédémontréedansplusieursétudescommetrèsricheencellules souchesmésenchymateuses.
Cescellulessouchesmultipotentesisoléesàpartirducordonombilicalontmontrédescapacitéd'auto renouvellement et de différentiation en une variété de type cellulaire ayant un potentiel pour des applications en therapie cellulaire, pas uniquement en tant que cellules différenciés mais également grâceàd'importantescapacitésimmunomodulatricesetrégulatricesdel'inflammation.
Cetravailprésenteledéveloppementdeméthodesetd'outils permettant untraitement optimisédes échantillonsdecordonombilicaletl'isolationd'explantsdetissusdegeléedeWhartonstandardisés.
Nousavonségalementétudiéplusieursméthodesaméliorantl'isolationdeMSCàpartirdecesexplants de tissus et développé un protocole de culture inͲvitro ne nécessitant pas l'ajout de sérum de veaux fœtaloulaprésenced'autresproduitsanimauxdérivésdanslebutdefaciliterdefuturesapplications cliniques.
Nous avons développé un protocole de cytométrie en flux multiparamétrique dédié à l'analyse de l'expressionde27antigènesetmarqueurdesurfaceprésentsurlesMSCdelageléedeWhartonetà l'étudedeseffetsdediversesconditionetévénementdurantlacultureinͲvitrosurleurphénotypede surface.
Lemêmeprotocolemultiparamétriqueaégalementétéutiliséafindecomparerl'expressiond'antigènes desurfacedesMSCisolésàpartirdelageléedeWhartonetdelamoelleosseuse,ouàdesfibroblastes.
Enfin, un protocole de différenciation ostéogénique a été appliqué avec succès aux MSC issues de la gelée de Wharton agrégées sur des granules d'hydroxyapatite fournissant ainsi un matériel autologue viable pour la réparation de manque osseux et spécialement développé pour la réparation de fente labioͲpalatines.
TableofContent
Acknowledgement... ...5 Abstract... ...7 Résumé:... ...8 Listoffigures... ...15 Listoftables... ...17 Listofabbreviations... ....18 1 Introduction... ...23 1.1 Stemcells... ...23 1.1.1 Stemcellssources...23 1.1.1.1 Embryonicstemcells...24 1.1.1.2 InducedPluripotentStemCells...26 1.1.1.3 Adultstemcells...27 1.1.1.4 AdultStemCelltypes...28 1.1.1.4.1 Bonemarrow...28 1.1.1.4.2 Adiposetissue...30 1.1.1.4.3 Adultstemcellsandtherapeuticapplication...30 1.1.2 Placenta,umbilicalcordandfoetalmembranes;roleandphysiologyofatransitional organ 31 1.1.2.1 Foetalmembranes...37 1.1.2.2 Thephysiologyoftheplacenta,roleinthefetoͲmaternalexchangeprocesses...38 1.1.2.2.1 Placentaasanimmunologicalbarrier...39 1.1.2.3 Theumbilicalcord...40 1.1.2.3.1 Embryogenesisoftheumbilicalcordandtissueorigin...40 1.1.3 Neonatalstemcells...43 1.1.3.1 Umbilicalcordblood...44 1.1.3.2 Umbilicalcordtissue...47 1.1.3.2.1 Physiologicroleoftheumbilicalcord...47 1.1.3.2.2 TheWharton’sJellyfunctionandmatrixcomposition...49 1.1.3.2.3 Cellsoftheumbilicalcord...51 1.1.4 Celltherapy,Regenerativemedicineandtissueengineering...54 1.1.4.1 Autologous,Allogeneicandcelltherapyapplication...55 1.1.4.2 Cellsfortherapeuticapplications...56 1.1.4.2.1 Safety,efficiencyandminimalmanipulationincelltherapy...561.2 Mesenchymalstemcells,adetailedanalysis...58 1.2.1 ThestemcellnatureofMSCs...60 1.2.2 ImmunophenotypeofMesenchymalStromalCells...61 1.2.3 MultipotentialityofMSCs...62 1.2.4 MSCandStromalSupportoftheStemCellNiche...63 1.2.5 ImmunomodulatorypropertiesofMSC...64 1.2.5.1 MSCsareinfluencedbythemicroenvironment...65 1.2.5.2 MSCandTlymphocytes...66 1.2.5.3 MSCandBlymphocytes...67 1.2.5.4 MSCandNaturalKillercells...68 1.2.5.5 MSCanddendriticcells...69 1.2.5.6 Secretionofsolublefactors...70 1.2.5.6.1 IndoleaminedioxygenaseorIDO...70 1.2.5.6.2 ProstaglandinE2orPGE2...71 1.2.5.6.3 TransformingGrowthFactorɴ1orTGFͲɴ1...71 1.2.5.6.4 TNFͲɲͲinducedprotein6orTSGͲ6...72 1.2.5.7 SecretionofproͲinflammatorymediatorsbyMSCs...74 1.2.5.8 MSCpolarizationintoproinflammatory(MSC1)orimmunosuppressive(MSC2) phenotype.... ...76 1.2.5.9 MSCsinhaematopoieticstemcelltransplant,interactionwithNKcells....78 1.3 DevelopmentofMSCtotheclinic...81 1.3.1 TheChoiceofMSCclinicalsource...81 1.3.1.1 AccessingMSCfrombonemarrow...82 1.3.1.2 AccessingMSCfromadiposetissue...83 1.3.1.3 AccessingMSCfromneonataltissues...85 1.3.1.3.1 UmbilicalCordMSCs...85 1.3.1.3.2 MesenchymalstemcellsfromtheWharton’sJelly...86 1.3.1.4 ComparisonofWJCstoAdultͲDerivedMSCs...87 1.3.1.5 Clinicalapplicationsofmesenchymalstemcells...88 1.3.1.5.1 ChallengesofMSCforcelltherapy,frombenchtobedside...91 1.3.1.5.2 SerumͲfree...92 1.4 Introductoryconclusions...94 1.5 Aimsandobjectivesofthisstudy...95 2 IsolationandexpansionofWharton'sJellyMSCinserumfreeconditions....97 2.1 Chapterintroduction...97 2.2 Materialandmethods...100
2.2.1 Collectionandprocessing...100 2.2.1.1 Wharton'sJellycollectionandprocessing...100 2.2.1.2 BoneMarrowcollectionandprocessing...103 2.2.1.3 Fibroblastscollectionandprocessing...103 2.2.2 MSCIsolation&Expansion...104 2.2.2.1 Wharton'sJellyMSCisolation...104 2.2.2.2 BoneMarrowMSCIsolation...104 2.2.2.3 Fibroblastsisolation...105 2.2.2.4 Passaging... 105 2.2.3 EvaluationofMSCisolationfromWJexplants...106 2.2.4 VariationofWJpiecesdensityduringIsolation...107 2.2.5 WJͲMSCisolationandexpansionusingsyntheticcoatingBDPureCoatECMMimetic Surfaces 108 2.2.6 WJͲMSCisolationandexpansioninhypoxicconditions...109 2.2.7 Cryopreservation...110 2.2.7.1 Cells... 110 2.2.7.2 WJslices... 111 2.2.7.3 WJpieces... 111 2.2.8 Cellthawingprocedure...111 2.2.8.1 Thawingofcellssuspension...111 2.2.8.2 ThawingofslicesandWJpieces...112 2.3 Results... ...113 2.3.1 Isolation,expansion,cryopreservationinSFcondition...113 2.3.1.1 EfficiencyofprocessingandIsolation...113 2.3.1.1.1 Processingmethod...113 2.3.1.1.2 Cordslicingholder...114 2.3.1.2 StatisticalcomparisonofUCslicingtooltomanualcutting...117 2.3.1.3 Umbilicalcordslicebacklightingdevice...119 2.3.1.4 IsolationofMSCfromFreshorfrozentissueinSForXSculturemedium....121 2.3.1.5 EvaluationofSFandXSmediuminMSCisolationfromWJexplants....122 2.3.1.5.1 FreshandFrozentissue...122 2.3.1.6 EffectofWJpiecesdensityduringisolationofMSC...124 2.3.1.7 WJͲMSCisolationandexpansionusingsyntheticcoatingBDPureCoatECMMimetic Surfaces 127 2.3.1.7.1 WJͲMSCIsolationusingMimeticSurfaces...127 2.3.1.7.2 WJͲMSCexpansionusingMimeticSurfaces...128
2.3.1.8 EffectofhypoxiccultureconditiononWJͲMSCIsolationandexpansion...129 2.3.1.8.1 WJͲMSCIsolationinhypoxiccondition...129 2.3.1.8.2 WJͲMSCExpansioninhypoxiccondition...130 2.3.1.9 ExpansionofWJͲMSCinserumͲfreeandserumcontainingmedium...131 2.4 DiscussiononWJͲMSCIsolationandExpansion...137 2.4.1 UmbilicalcordMSC...137 2.4.2 DefinemethodsfortheproductionofUCͲMSCforcelltherapy...139 2.4.3 DefinedmediafortheIsolationofWJͲMSC...140 2.4.4 IsolationofMSCfromWJaftercryopreservation...142 2.4.5 InfluenceofWJexplantdensityonMSCisolation...143 2.4.6 BDMimeticFibronectinsyntheticcoatingasreplacementofhumanderivedcollagenIͲIII 143 2.4.7 HypoxicculturetoincreaseefficiencyofMSCisolationandexpansion...144 2.4.8 ExpansionofWJMSCindefinedSFmedium....145 2.5 Conclusion... .147 3 FlowcytometryanalysisofWJͲMSC...149 3.1 Chapterintroduction...149 3.2 Materialandmethods...151 3.2.1 Cellharvestingandstaining...151 3.2.2 Compensationofemissionspectraloverlap...154 3.2.2.1 Specificnoteondeterminationofthepositivecellpopulation...154 3.2.2.1.1 Unstainedcontrol...154 3.2.2.1.2 Isotypecontrols...154 3.2.2.1.3 FMOcontrols...155 3.2.3 SampleProcessing...156 3.2.4 FACSdatatreatment...161 3.2.4.1 Gatingmethodology...161 3.2.4.2 Statisticaltreatmentofdata...168 3.3 Descriptionofanalyzedsurfaceepitopesandmarkers....169 3.4 Resultsanddiscussion...185 3.4.1 Introductorynote...185 3.4.2 EffectofpassagenumberonWharton'sJellysurfaceantigensexpression...187 3.4.2.1 ComparisonoflowandhighpassageinXSculturemedium...187 3.4.2.2 ComparisonoflowandhighpassageinSFculturemedium...189 3.4.2.3 ComparisonoflowandhighpassageinXSorSFculturemedium...194 3.4.2.4 FinalstatementonWJͲMSCsurfacemarkerexpressionatlowandhighpassages....197
3.4.1 EffectoffreezingonWharton'sJellysurfaceantigensexpression...198 3.4.1.1 ComparisonofWJͲMSC,Freshorafterafreeze/thawcycleinXScultureandfreezing medium 198 3.4.1.2 ComparisonofWJͲMSCculturedinSFmediumbeforeorafterafreeze/thawcycle.204 3.4.1.3 ComparisonofWJͲMSC,Freshorafterafreeze/thawcycleinSForXSculture...207 3.4.1.4 FinalstatementonsurfaceantigensexpressionofWJͲMSC,freshorafterafreezeͲ thawcycle.... ...210 3.4.2 ComparisonofWJͲMSCculturedinSerumͲFreeorXenoͲSerumcontainingmedium....211 3.4.2.1 FinalstatementonantigensexpressionofWJͲMSCinSForXScultureconditions....220 3.4.3 ComparisonofsurfaceantigenexpressionofWJͲMSC,BMͲMSCandFibroblastscultured inSFcondition.... ..221 3.4.3.1 ComparisonofBMͲMSCandFibroblasts....221 3.4.3.2 ComparisonofWJͲMSCandFibroblasts....229 3.4.3.3 ComparisonofWJͲMSCandBMͲMSC....241 3.4.3.4 FinalstatementonsurfaceantigenexpressionofWJͲMSC,BMͲMSCandFibroblastsin SFcondition... ...251 3.5 DiscussiononsurfaceantigenexpressionofMSC...253 3.6 Conclusion... .257 4 ExampleofaclinicaldevelopmentofhumanWharton'sJellyMSCtowardosteogenicbone implantation... ...259 4.1 ChapterIntroduction...259 4.1.1 Embryonicdevelopmentofbone...259 4.1.2 Bonedamageanddefects...261 4.1.3 Bonetissueengineering...262 4.1.4 MSCdifferentiationintobonetissueforcleftlipandpalaterepair....262 4.1.5 UseofBMPͲ2totriggerMSCdifferentiationintobonetissue....263 4.2 Materialsandmethods...264 4.2.1 ConstructsofWhartonJelly–DerivedMesenchymalStromalCells,Fibrin,and Hydroxyapatite... ..264 4.2.2 Controlandosteogenicdifferentiationmedium...264 4.2.3 Experimentalplan...264 4.2.4 Samplepreparation...265 4.2.5 Histologicalstaining...265 4.2.6 Observation... 265 4.2.7 Collaborations...265 4.3 Results... ...266 4.3.1 Histologicalanalysisfollowingosteogenicdifferentiation...266
4.3.2 Samplesafter21daysofdifferentiation...266 4.3.2.1 CultureofcellconstructsincontrolconditionCͲDMEM...266 4.3.2.2 CultureofcellconstructsindifferentiationmediumFͲDMEM...268 4.3.2.3 CultureofcellconstructsindifferentiationmediumFͲDMEM+BMPͲ2...270 4.3.3 Samplesafter42daysofdifferentiation...272 4.3.3.1 CultureofcellconstructsincontrolconditionCͲDMEM...272 4.3.3.2 CultureofcellconstructsindifferentiationmediumFͲDMEM...274 4.3.3.3 CultureofcellconstructsindifferentiationmediumFͲDMEM+BMPͲ2...276 4.4 Discussion... ..278 4.5 Conclusion... .281 5 Summary... ...282 6 Furtherdevelopments... 283 7 Bibliography:... ...284 8 Communications... 318 9 Appendixes... ...319 Appendix1:Noted’informationetfichedeconsentement...319 Appendix2:Formulaired'identificationdeséchantillons....320
Listoffigures
Figure1:SchematicofhEScelllinesfromhumanblastocyst...25 Figure2:Blastocystschemaandpictureonthefifthday...32 Figure3:Hatchingoftheembryos...32 Figure4:Implantation,6thͲ7thand7thͲ8thdays...33 Figure5:Implantation,8thand9thdays...33 Figure6:Implantation,9thͲ10thand10thͲ11thdays...34 Figure7:Implantation,9thͲ10thday,LacunarstageandPrimaryvillus,11thͲ13thday...34 Figure8:Secondary,tertiaryandfreevilliatthe16thand21stdayandattheendofthe4thmonth...35 Figure9:Schematicdiagramoftheplacentaataroundthefourthmonthinasagittalsection. (www.embryology.ch)... ...36 Figure10:Placentafromthematernalandfetalside....36 Figure11:Foetalmembranesatthe8thand12thweeks....37 Figure12:UteroͲplacentalcirculationsystem...38 Figure13:Bodystalkaroundthe3rdweekandformationoftheumbilicalcordaroundthe3.5thweek. (www.embryology.ch)... ...42 Figure14:Umbilicalcordandumbilicalvesicleinthechorioniccavity....42 Figure15:Umbilicalcorddevelopment....42 Figure16:UmbilicalcordbloodcollectionafterCaesareandelivery....46 Figure17:FoetusinͲuterobetween5and6months....47 Figure18:Umbilicalcordstructureatbirth....48 Figure19:DiagramofWharton'sJellyMacromolecularcomposition....50 Figure20:Diagramoftheumbilicalcordandthedifferentstem/progenitorcellsderivedfromthebloodandtissue. ... ...53 Figure21:MesenchymalStemCellphenotypeduringinͲvitroculture....59 Figure22:MSCspresentmultipotentdifferentiationcapability....62 Figure23:StromalcellsinthehaematopoieticstemͲcellniche....63 Figure24:PossiblemechanismsoftheinteractionsbetweenMSCsandcellsoftheinnateandadaptiveimmune systems.... ...73 Figure25:PossiblemechanismsinvolvedinMSCproinflammatoryorimmunosuppressivepolarization....77 Figure26:Thevarioussteps,processandexperimentsperformedduringWJͲMSCevaluation....99 Figure27:UmbilicalcordprocessingandWharton'sJellyisolation....102 Figure28:BiopsypunchesusedforWJisolationfromumbilicalcordslices....113 Figure29:Schematicsoftheinitialversionofthecordholder....114 Figure30:Secondversionofthecordslicingholder....115 Figure31:Transversalslicingofumbilicalcordsampleusingtheholder....116 Figure32:Comparisonoftheaverageweightoftheslicesobtainedfromeachsamplewhencutmanuallyorusing theholder....118 Figure33:Prototypeoftheumbilicalcordslicebacklightingdevice....120 Figure34:Secondversionofthebacklightingdevice....120 Figure35:IsolationofMSCfromoneWharton'sjellytissueexplant....121 Figure36:AveragedurationofWJͲMSCisolationinvariousconditions....123 Figure37:ComparativeexpansionofWJͲMSCinSFSPEͲIVorDMEM+10%FBSmedium....134 Figure38:ComparativeexpansionofWJͲMSCisolatedfromfreshorfrozentissueinSFSPEͲIVorXSDMEM+10% FBS.... ...135 Figure39:CumulativepassagingtimeduringWJͲMSCexpansioninSFSPEͲIVorXSDMEM+10%FBSmedium....136 Figure40:BDLyseͲWashAssistantusedtoremoveanyunboundantibodies....156 Figure41:LightpathwaythroughtheBDFACSCANTOIIflowcytometer....157 Figure42:BDFACSCantoIIcytometerlaseropticalpathway....158 Figure43:DiagramofBDFACSCantoIIcytometerflowcell....158Figure45:DiagramofBDOctagonfluorescentdetectorarray....159 Figure46:BDFACSCantoII4Ͳ2Ͳ2octagonopticaldetectorarrayfrom488Ͳnmbluelaser....160 Figure47:BDFACSCantoII4Ͳ2Ͳ2octagonopticaldetectorarrayfrom633Ͳnmredand405Ͳnmvioletlaser....161 Figure48:Gatingmethodologyfordeadcellexclusion....162 Figure49:ExampleofstainingandMSCsubpopulationobservedonbiͲparametricdotͲplotinthetube3,oneofthe 6tubesoftheanalysispanel(Table14)....165 Figure50:AnalysisofmonoͲparametrichistogramsstainingobtainedonWJͲMSCinoneoftheanalysistube(Tube 3).... ...167 Figure51:Exampleofpopulationhierarchyduringflowcytometryanalysis(Tube3)....168 Figure52:Surfaceexpressionofthe27antigenscomposingtheanalysispanelonFibroblast,BMͲMSCandSForXS WJͲMSC.... ...186 Figure53:VariationofCD34andCD31surfaceexpressiononWJͲMSCculturedinXSmediumatlowandhigh passages.... ...187 Figure54:AntigenspresentingvariationofsurfaceexpressiononWJͲMSCculturedinSFmediumatlowandhigh passages.... ...189 Figure55:AntigenspresentingvariationofsurfaceexpressiononWJͲMSCculturedinSForXSmediumatlowand highpassages.... ...194 Figure56:SurfaceantigenspresentingdifferencesofexpressiononWJͲMSCculturedinXSmediumbeforeorafter afreezethawcycle....198 Figure57:SurfaceantigenspresentingdifferencesofexpressiononWJͲMSCculturedinSFmediumbeforeorafter afreezethawcycle....204 Figure58:AntigenspresentingdifferencesofsurfaceexpressiononWJͲMSCculturedinXSorSFmediumbeforeor afterafreezethawcycle.... ...207 Figure59:SurfaceantigenspresentingdifferencesofexpressiononWJͲMSCculturedinXSorSFmedium....212 Figure60:AntigenspresentingdifferencesofsurfaceexpressionbetweenBMͲMSCandFibroblastculturedinSF medium.... ...222 Figure61:AntigenspresentingdifferencesofsurfaceexpressionbetweenWJͲMSCandFibroblastculturedinSF medium.... ...230 Figure62:AntigenspresentingdifferencesofsurfaceexpressionbetweenWJͲMSCandBMͲMSCculturedinSF medium.... ...242 Figure63:Surfaceexpressionofthe27antigenscomposingtheanalysispanelonFibroblast,BMͲMSCandSForXS culturedWJͲMSC.... ...255 Figure64:DifferentiationofmesenchymalprogenitorcellsisregulatedbyWntsignaling....260 Figure65:Endochondralboneformationistiedtocartilagedevelopment....261 Figure66:Sample13E1344after21daysinthecontrolmedium....267 Figure67:Sample13E1345after21daysinthedifferentiationmediumFͲDMEM....269 Figure68:Sample12E1349after21daysofdifferentiationinFͲDMEM+BMPͲ2medium....271 Figure69:Sample13E1641after42daysinthecontrolmedium....273 Figure70:Sample13E1642after42daysinthedifferentiationmediumFͲDMEM....275 Figure71:Sample12E1643after42daysofdifferentiationinFͲDMEM+BMPͲ2medium....277 Figure72:Rangeofosteogenicdifferentiationachievedduringourexperiment....280
Listoftables
Table1:MacromolecularcompositionoftheextracellularmatrixofWharton'sJelly....50 Table2:GradingtableusedformonitoringofWJͲMSCisolationculture....106 Table3:Slicewetweightcomparisonwhenproducedusingcuttingdeviceorbymanualtechnique....117 Table4:NormalizedMSCnumberisolatedfromdifferentsamplewhenWJbiopsyareseededatvariousdensity.124 Table5:TotalMSCnumberisolatedfromdifferentsamplewhenWJbiopsyareseededatvariousdensity....124 Table6:Wharton'sJellyseedingdensitypresentingstatisticallysignificantdifferences(cellperWJnumber)...125 Table7:Wharton'sJellyseedingdensitypresentingstatisticallysignificantdifferences(totalcellnumber)...125 Table8:EfficiencyofBDMimeticcoatingtosupportWJͲMSCisolationcomparedtohCollagen....127 Table9:AverageyieldonWJͲMSCculturedonhumanCollagenandBDmimeticfibronectinorcollagencoatingat P1(n=4).... ...128 Table10:TotalWJͲMSCnumberharvestedfromtissueexplantculturedinhypoxicornormoxicconditions(n=6). ... ...129 Table11:TotalWJͲMSCnumberharvestedafterexpansioninhypoxicornormoxiccondition(n=6)....130 Table12:ComparisonoftheaveragecellnumberandtimeateachpassageswhenWJͲMScareculturedinSFSPEͲ IVorXSDMEM+10%FBSmedium....132 Table13:Listofantibodies(withcorrespondingisotype)orstainingreagentandtheirrespectivesupplierand reference,usedforMSCcharacterization....152 Table14:Descriptionofthe6differentcombinationsofantibodiescoupledwiththeirfluorochromesandtheir respectiveexcitationwavelengthanddetectionchannel....153 Table15:BDCantoIIopticalfilterconfiguration....160 Table16:CorrelatedvariationofCD34andCD31expressiononWJͲMSCculturedinXSmediumatlowandhigh passages.... ...188 Table17:SummaryofthedifferencesinsurfaceantigensexpressionobservedbetweenWJͲMSCatloworhigh passages.... ...197 Table18:Summaryofthedifferencesinsurfaceantigensexpressionobservedbeforeoraftercryopreservation.210 Table19:VariationofCD10surfaceexpressiononfibroblastsculturedinSForXSmedium....217 Table20:VariationofD7ͲFibsurfaceexpressiononfibroblastsculturedinSForXSmedium....218 Table21:VariationofCD15surfaceexpressiononfibroblastsculturedinSForXSmedium....219 Tableau22:SummaryofthedifferencesinsurfaceantigensexpressionobservedonWJͲMSCculturedinSForXS medium... ...220 Table23:VariationofCD34surfaceexpressiononfibroblastsculturedinSForXSmedium....223 Table24:VariationofHLAͲGsurfaceexpressiononfibroblastsculturedinSForXSmedium....224 Table25:VariationofCD14surfaceexpressiononfibroblastsculturedinSForXSmedium....225 Table26:VariationofCD63surfaceexpressiononfibroblastsculturedinSForXSmedium....226 Table27:VariationofCD56surfaceexpressiononfibroblastsculturedinSForXSmedium....227 Table28:VariationofCD133surfaceexpressiononfibroblastsculturedinSForXSmedium....231 Table29:VariationofCD34surfaceexpressiononfibroblastsculturedinSForXSmedium....232 Table30:VariationofCD45surfaceexpressiononfibroblastsculturedinSForXSmedium....233 Table31:VariationofHLAͲGsurfaceexpressiononfibroblastsculturedinSForXSmedium....234 Table32:VariationofCD63surfaceexpressiononfibroblastsculturedinSForXSmedium....236 Table33:VariationofCD56surfaceexpressiononfibroblastsculturedinSForXSmedium....237 Table34:VariationofCD146surfaceexpressiononfibroblastsculturedinSForXSmedium....238 Table35:VariationofCD15surfaceexpressiononfibroblastsculturedinSForXSmedium....239 Table36:SummaryofthedifferencesinsurfaceantigensexpressionobservedbetweenWharton'sJellyMSC,Bone MarrowMSCandFibroblastsculturedinSFmedium....252 Table37:Expressionaldiagramcomparingsurfaceexpressionofthe27antigensonFibroblast,BMͲMSCandSFor XSculturedWJͲMSC.... ...256Listofabbreviations
7AAD 7ͲAminoactinomycinD AF647 AlexaFluor647 AGM AortaͲGonadMesonephros ALCAM ActivatedLeukocyteCellAdhesionMolecule AML AcuteMyeloidLeukemia APC Allophycocyanin APCͲH7 AllophycocyaninHilite7 APCs AntigenͲPresentingCells ASC AdultStemCells ATͲMSC AdiposeTissuederivedMSC BM Bonemarrow BMPs BoneMorphogeneticProteins CALLA CommonAcuteLymphoblasticLeukemiaAntigen CFUͲF ColonyFormingUnitͲFibroblasts CFUͲGEMM ColonyFormingUnitͲGranulocyteErythrocyteMonocyteMegakaryocyte CFUͲGM ColonyFormingGranulocyteMacrophages COX Cyclooxygenase CPDͲA CitratePhosphateDextroseAdenine CST CytometerSetupandTracking CTL CytotoxicTLymphocytes DCs DendriticCells DMEM Dulbecco’sModifiedEagle’sMedium DMSO DimethylSulphoxide DͲPBS Dulbecco’sPhosphateͲBufferedSaline ECM ExtraCellularMatrix EEM ExtraͲEmbryonicMesoblast EPC EndothelialProgenitorCellsEPC EndothelialProgenitorCell FBS FoetalBovineSerum FCS FoetalCalfSerum FGFͲ2 FibroblastGrowthFactorͲ2 FGFR FibroblastGrowthFactorReceptor FITC FluoresceinIsothiocyanate FMO FluorescenceMinusOne FSC Forwardscatter GMP GoodManufacturingPractice GPA GlycophorinA GPI Glycophosphatidylinositol GVHD GraftVersusHostDisease HCELL HematopoieticCellEͲselectin/LͲselectinLigand hESCs HumansEmbryonicStemCells HIV HumanImmunodeficiencyVirus HLA HumanLeukocyteAntigen HSC HaematopoieticStemCell HSCT HaematopoieticStemCellTransplant HUVECs HumanUmbilicalVeinEndothelialCells ICAM IntercellularAdhesionMolecule IDO Indoleamine2,3Ͳdioxygenase IFN Interferon Ig Immunoglobulin IL Interleukins iNOS inducibleNitricͲOxidesynthase iPSC Inducedpluripotentstemcells ISCT InternationalSocietyforCellularTherapy ITIMs ImmunoreceptorTyrosineͲbasedInhibitoryMotifs
LCA LeukocyteCommonAntigen LED LightEmittingDiode LFA LymphocyteFunctionalAntigen LGL GranularLymphocyteLeukemias LIF LeukemiaInhibitoryFactor LPS Lipopolysaccharide MAPCs MultipotentAdultProgenitorCells MCAM MelanomaCellAdhesionMolecule MHC MajorHistocompatibilityComplex MLR MixedLymphocyteReactions MSCs MesenchymalStemCells NK NaturalKiller OctͲ4 Octamer4 OI OsteogenesisImperfecta PBS PhosphateͲBufferedSaline PDGFͲBB PlateletͲDerivedGrowthFactorͲBB PE Phycoerythrin PEͲCy7 PhycoerythrinCyanin7 PerCPͲCy5.5 PeridininͲChlorophyllͲProteinͲCyanin5.5 PGEͲ2 ProstaglandinE2 PLA ProcessedLipoaspirateCells PMTͲV PhotomultiplierTubeVoltage PSA PenicillinͲStreptomycinͲAmphotericinB PTP ProteinTyrosinePhosphatase SEM StandardErrorofMean SF SerumͲFree SMA SmoothMuscleActin SoxͲ2 SRY(SexDeterminingRegionY)Ͳbox2
SSC SideScatter SSEAͲ1 StageͲSpecificEmbryonicAntigen1 TGF TransformingGrowthFactor TLR TollLikeReceptors TNF TumorNecrosisFactor TNFAIP6 TNFͲɲͲinducedprotein6 Tregs TͲregulatorycells UC UmbilicalCord UCB UmbilicalCordBlood VCAM VascularCellAdhesionMolecules VLA VeryLateAntigen WJ Wharton’sjelly WJC Wharton’sjellyCell WJͲMSCs Wharton’sjellyderivedMSCs XS XenoͲSerum
Chapter1
Introduction
1 Introduction
1.1 Stemcells
Stemcellsareprimordial,undifferentiatedcellswhichhavenottakenontheidentityofanyspecificcell type and can be induced to differentiate and give rise to one or more specialized cell type, allowing them to act as a repair system for the body, replenishing other cells through the lifetime of the organism. Alternatively some of the progeny arising from the stem cell division will retain stem cell properties, allowing through this selfͲrenewal capacity the maintenance of a constant stem cell pool (Smith,2001;Till&McCulloch,1961;Weissman,Anderson,&Gage,2001).
1.1.1 Stemcellssources
Mammalian stem cells are often primarily classified from their sources, and two main classes of stem cellsareusuallyrecognizedthroughthescientificcommunity.Embryonicstemcells,harvestedfromthe blastocystofanembryo,appeartogiverisetoallspecializedcelltypes,withtheexceptionofasetof extraͲembryonic cells giving them pluripotent capacity. Adult stem cells on the other hand can be sourcedfromvarioustissuesofthehumanbody,presenttovaryingdegreesofrestrictiontoaparticular lineageandeaseofaccess(Watt&Contreras,2005).
Recently, exploration of the potency of stem cells derived from the placenta, foetal membranes, umbilicalcordandcordblood,tissuesusuallydiscardedafterbirth,hascalledforthedefinitionofathird source of stem cells, as indeed, their means of access, potency of differentiation, immunonaivity and tolerance.Finallypotentialscaleofcollectionworldwidewithoutanyethicalissues,setneonatalstem cellsapartbothfromtheembryonicandadultstemcellsgroups(Forraz&McGuckin,2011).
1.1.1.1 Embryonicstemcells HumansEmbryonicStemCells(hESCs)werefirstderivedin1998(Thomson,1998),bytheisolationand expansionincultureofthe30to34cellsoftheinnercellmassofanearly4to5daysblastocyst,they areusuallyderivedfromembryosdestinedfordestructionataninͲvitrofertilizationclinic. TheisolationandcultureofhESCsallowedthepossibilitytostudyhumandevelopment,usually inaccessibleinͲvivoanddevelopnewapproachesinordertotreatalargerangeofdiseases. SeveralcharacteristicsdefineESCs;theycanselfͲrenewandthusproliferateextensivelyinculturewhile maintaining an undifferentiated state and a normal karyotype in the proper culture conditions. FurthermoreESCsarebroadlypluripotentandretainalargedevelopmentalpotential,bybeingableto differentiate into cells from ectodermal, endodermal and mesodermal germ layers (Amit et al., 2000; Hoffman&Carpenter,2005),finallytheyalsomaintainahighlevelofOctͲ4expressionandtelomerase activity.
ESCs having the capacity to differentiate into clinically relevant cell types, such as dopaminergic neurons,cardiomyocytesorinsulinͲproducingbetacells,thereisahugeinterestinusingthosecellsfor both research purpose and regenerative medicine or transplantation. Both will however require a precise cell control and several experimental approaches are investigating the expansion and differentiationpotentialtospecificlineages.
The great level of plasticity of ESCs is both the main advantage and a major limitation for a potential clinicaluseinregenerativemedicineapplication.Indeedoneoftheassaydefiningthecharacteristicsof anESClineisthevalidationofitscapacitytoproduceembryonicbodiesinͲvitroandteratomas,theESC
inͲvivocapabilitytoproducecelltypesrepresentingallthreeEGlayersinadisorganizedform(Cowanet al., 2004). One of the major challenges to overcome will be the essential capacity to exclude any
undifferentiatedcellsandtoinsuretheabilitytoproduceapurepopulationofspecificallydifferentiated cellsbeforetheuseinclinicofanycellularproductderivedfromEScells.
CultureconditionsareanotherlimitationasESCswereoriginallyestablishedandmaintainedbythecoͲ culturewithmitoticallyinactivatedembryonicfibroblastfeederlayersand/orgrowninculturemedium supplemented with animal serum. The use of xenogeneic components raises the question of the infectionwithnonhumanpathogensandsincemanyofthecurrentlyavailablecelllinesofhumanESCs wereexposedtheyareunlikelytobeusedinclinicaltherapies.
AmainissuethatmustbeaddressedbeforetheuseofESCsintherapyisimmunerejectionbasedon mismatchesatgenetichistocompatibilityloci.Indeedduetotheirnaivenatureitwasfirstassumedthat theywouldnotneedimmunosuppressionupontransplant,butunfortunatelyithasbeenshownthatES cellsandtheirderivativescanbeinducedtoexpresshighlevelsofMHCclassIantigens(HLAͲAandHLAͲ B), therefore any ES cell based product is likely to be subjected to rejection by an immunocompetent host(Drukkeretal.,2002). Itisalsoimportanttomentionthattheuseofthesecellsisperceivedascontroversialbysomegroups andmajorethicalandpoliticalissuesarestilltoberesolved. StillthestudyofhESCsisconsideredbysometoremainessentialsinceitmaybringknowledgeonthe capacityofcellstoretainoracquireapluripotentstateandsuchabasicunderstandinghasimplications thatgoesfarbeyondthepotentialuseofEScellsfortransplantationandmayleadtothediscoveryof methodshelpingtissuetoregenerate. TodatethereseemstobenocurrentclinicaltreatmentbasedonESCandonlyfewmodestpublished workswithsuccessonanimalmodels. Figure1:SchematicofhEScelllinesfromhumanblastocyst. Theinnercellmasscanberemovedandculturedoninactivatedmouseembryonicfeedercells,resulting afterseveralpassageintheformationofhESCline.FromVeeck,L.andZaninovic,N.AnAtlasofHuman Blastocysts,Parthenon,BocaRaton,Florida2003.(Hoffman&Carpenter,2005).
1.1.1.2 InducedPluripotentStemCells
Induced pluripotent stem cells (iPSC) are a relatively new addition to the world of stem cells as they were introduced in 2006 in a research article where Shinya Yamanaka and colleagues described the reprogrammingofmousefibroblastsintoapluripotentcellasusingtheretroviraltransductionoffour genes(OctͲ4,SoxͲ2,klf4andcͲMyc).
The somatic cells were able to express characteristics of morphology, growth rate or gene expression similartomouseembryonicstemcells(mESC)andinadditiontotheformationofembryoidbodiesthey couldalsobedifferentiatedintocelltypesfromallthreegermlayers(Takahashi&Yamanaka,2006). TheprocessforIPSgenerationwasrefinedbyYamanaka'sgroupsin2007(Takahashietal.,2007)and muchprogresshasbeenmadesincewiththegenerationofIPSfromhumancellsortheuseoflentiviral ratherthanretroviraltransfectionallowingtransductionofnonͲdividingcells(Parketal.,2008;Yuetal., 2007;Yu,Hu,SmugaͲOtto,Tian,&Stewart,2009) TheprogressonIPSgenerationopenspossibilitiesforstemcellsderivedfromthepatient'sowncellsfor potentialdiagnosticresearchandperhapsclinicaluse. Butalthoughtheydisplayimportantpotentialitisimportanttoobservethatthecurrentreprogramming efficiencyofIPSremainsverylowatapproximately0.1%,thattheysharemajorsafetyissueswithESCas theytooreadilyformteratomaswhenimplantedinimmunodeficientmice,andthattheveryprocessfor IPS generation is at the time considered less safe and more tumorigenic than ESC as several genes involvedareknownoncogenes.
IPSC also raise concerns regarding their immunogenicity as, although reports are conflicting, it seems theyareabletoproduceastrongenoughimmuneresponsetocauserejectionwhileequivalentESCare tolerated(Arakietal.,2013;T.Zhao,Zhang,Rong,&Xu,2011)
Although the possibility of patient specific cell therapy treatment offered by IPSC is seducing, it is importanttomentionthatmuchworkisneededtoovercomethetechnicalandsafetyconcernsraised by these cells before clinical applications (Okano et al., 2013) and the IPS ageing through accelerated reductionofthetelomereofanalreadymaturecellisthemajorlimitationasitcouldpotentiallyspeed mutagenesis.
Althoughitmayseemeasiertousecellspresentingawidercapacityfordifferentiation,thechallenges of eliminating residual undifferentiated stem cells and controlling lineage specific differentiation may greatlydelayasafeclinicalapplicationforEmbryonicStemCellsorIPSC.Inmanycases,AdultStemCells andNeonatalStemCellsmayprovideaneasierandmoredirectroutetocelltherapyandregenerative medicine. 1.1.1.3 Adultstemcells Adultstemcellsarefoundinmature tissues,abletoselfͲrenewanddifferentiateintomoreadvanced celltypes.Firstthoughttobeonlylocatedinalimitednumberoforgan,andonlyabletodifferentiate intothephenotypespresentsinthetissuesoforigin,manyrecentstudieshavedemonstrateditisnot only likely that each organ or tissue has one somatic stem cell but also a greater potential than was originallypredicted.
Adult stem cells have now been identified in human bone marrow (Edwards, 2004), blood (Ogawa, 1993), endothelial cells (Asahara, 1997),neural (Doetsch, 2003; Song, 2003), fat (Zuk et al., 2002a) Hepatic tissue (Schmelzer, Wauthier, & Reid, 2006; Suzuki et al., 2002; Tosh & Strain, 2005), muscle (Zammit, Partridge, & YablonkaͲReuveni, 2006; Zheng et al., 2007), pancreas (Zulewski, Abraham, & Gerlach,2001)andcardiac(Beltramietal.,2003;Ohetal.,2003). Itseemsnowverylikelythateachtissueororganhassomaticstemcellsintheadult;animmaturecell groupcapableoftissuerepair,maintainingtissuehomeostasis.Forexampleinmaintainingbloodorskin throughlife,andrepairinanichespecificcontrolledmicroenvironment(L.Li&Xie,2005). AlthoughlessextendedthanforESC,thesepopulationsofadultstemcellspresentahighcapabilityfor selfͲrenewalinculture,haveinsomecasesbeenfoundtoexpresstelomeraseandnotbeingsubjected to replicative senescence. They are generally classified as multipotent, able to generate various cells fromthesametissue.Neuralstemcellscanforexampleyieldneurons,astrocytesandoligodendrocytes; cardiac stem cells can differentiate into cardiomyocytes, smooth muscle, and endothelial cells while muscle derived stem cells yield skeletal muscle and can be differentiated into chondrocytes. These apparently lineage restricted stem cells seem to be, unlike ES cells, non tumorigenic, and thus could potentiallybeusedforcelltherapyproductswithorwithoutpreliminarydifferentiation.
Moreover, it seems possible for some lineage specific ASC to be capable of a larger plasticity than expectedfromtheirtissueorigin.Forexampleitseemthathepaticstemcellscanbeinducedintocells oftheendocrinepancreas(W.ͲC.Li,Horb,Tosh,&Slack,2005;Yangetal.,2002;Zalzman,AnkerͲKitai,& Efrat,2005).Thistypeofswitchbetweenrelatedcelllineagesmayturnouttobeeasierthantoinduce thefulldevelopmentalprogramstartingfromaveryprimitiveprecursorlikeEScells.
Moreover we must not forget that thanks to bone marrow transplant we now have more than 3 decades of clinical background demonstrating the safe use of adult stem cells for cell therapy applications.
Someofthe different typesoftissuespecificstem cellsthatarewelldocumented torespondtolocal tissuedamageandcontributetotissueregenerationinadults: Ͳ Livercellsthatproliferatefollowingpartialhepatectomy Ͳ Haematopoieticstemcellsthatreconstitutethebloodfollowingirradiation, Ͳ Satellitecellsthatrepairdamagesskeletalmuscles Ͳ Keratinocyteprecursorsthatparticipateinwoundhealing 1.1.1.4 AdultStemCelltypes
Although adult stem cells may ultimately be derived from almost every tissue of the body, some cell sourcesaremorelikelytocontributetoregenerativemedicine,basedoneaseofisolation,availabilityor potency. 1.1.1.4.1 Bonemarrow Bonemarrowisahaematopoietictissuefoundmainlyintheflatbones,suchasthepelvic,sternum,rib, vertebrae,andinthecancellousmaterialinthehollowcentralportionoflongbonessuchasthefemur andhumerus.
This haematopoietic tissue is physically supported and physiologically maintained by the trabecular bone and the bone marrow stroma constituted of reticular cells, adipocytes, osteocytes, vascular endotheliumandextracellularmatrix.
Associated with the blood vessels, the bone marrow forms a haematopoietic inductive microenvironment,siteofproductionofallcellsinvolvedinhaematopoiesisandthedailyproductionof fivebillionbloodcellsinadults. Bonemarrowisahighlyvascularisedtissueandthesinusoidalvasculatureinwhichtheendothelialcells arenotencapsulatedwithothercelltypes,formsahighlypermissivestructurefortheimmigrationand emigrationofbonemarrowcells. Bonemarrowis,inadults,amajorreservoirofmultipotentstemcells,whichcangiverisetomostofthe adultcelltype(Stocum,2001)
Thankstoitseaseofisolation,thistissuehasbeenthoroughlyinvestigatedandseveralgroupofstem cellshavebeenidentifiedwithinbonemarrow,haematopoieticstemcells,(Ogawa,1993)differentiating intobloodcellsandtheendothelialprogenitorcells(EPC)(Asahara,1997;Kocheretal.,2001),butalso MesenchymalStemCells(MSCs)(Y.Jiangetal.,2002;Pittengeretal.,1999).
Haematopoieticstemcellsareentirelyresponsibleforthedevelopment,maintenanceandregeneration of the blood forming tissue, giving rise to all red and white blood cell types, for the entire life of the organism(I.L.Weissman,2000).
Sincetheycanreconstituteandrestorethehaematopoieticsystemofamyeloablatedhost,theyhave beenusedforseveraldecadesinthetreatmentofhematologicdisorders,startingin1945,whenthey were first used to treat lethally irradiated civilian populations (Gengozian & Makinodan, 1958) and several studies have since indicated the existence of highly plastic stem cells, known as bone marrow derivedstemcells,(Leoneetal.,2005),exhibitingaverybroaddifferentiationpotentialinseveralnonͲ haematopoieticorgans.
BonemarrowisamongstothertissuesasourceofMesenchymalStemCells(MSC),andthefirstwere these nonͲhaematopoietic, structural components of bone marrow, where identified as supporting haematopoiesisthroughextracellularmatrixcomponents,releaseofcytokinesandgrowthfactors,but havealsobeenprovedabletogeneratebone,cartilage,fatandfibrousconnectivetissue.
DifferentgroupsofcellsthatinitiallycoͲpurifywithMSCfromthepostnatalmarrow(andotherorgans) of mice, rat and humans, growing as adherent cells inͲvitro has been identify and referred to as multipotentadultprogenitorcells(MAPCs).UnlikeMSCtheycanbeculturedinarelativelynutrientpoor mediumandinducedintomyoblasts,hepatocytesandneuraltissue(Y.Jiangetal.,2002).
Endothelialprogenitorcells(EPCs)areanothergroupofcellsfoundwithinthebonemarrowHSC,MSC and other tissues like fat, cord blood or circulating blood, that do not yet show endothelial characteristics but can be differentiated into endothelial cells. HSC derived EPC are the most characterized and reside in the BMSC niche and released upon mobilization through cytokines synthesizedbyischemictissue(suchasvascularendothelialgrowthfactorVEFGorstromalcellderived factorͲ1SDFͲ1)(Leoneetal.,2005).
1.1.1.4.2 Adiposetissue
WhileMSCwerefirstdescribedandisolatedinbonemarrow,itisnowverywellestablishedthatthey arealsopresentinmanyothertissuesinadults,oneofthembeingtheadiposetissue(DeUgarteetal., 2003;Zuketal.,2002a).
TheadvantagetocollectMSCfromadiposetissueinsteadofbonemarrowareveryclear,collectedwith only local anaesthesia, lipoaspiration is a routinely executed procedure associated with only minimal risksanddiscomfort(althoughsomemaydisagree).
Afterharvesting,thelipoaspirateistreatedusingcollagenase,thenfollowingcentrifugation,astromal vascular fraction, similar to the MNC fraction of the BM, can be collected, while the adipocyte containing fraction is removed thanks to it’s high content of fatty acids. After plating of the stromal vascular fraction, a plastic adherent CFUͲF forming cell population can be isolated, originally named Processed Lipoaspirate cells (PLA) (Katz, Tholpady, Tholpady, Shang, & Ogle, 2005; Zuk et al., 2001, 2002a).
TheywereshowntohavebothahighpotentialforinͲvitroexpansionanddifferentiation,intocelltypes notonlyderivedfromthemesodermallineage,asbone,cartilage,fat,muscle,heart,bloodvesselsand nerves could be obtained (Strem et al., 2005) and while PLA cells appear quite similar to BM derived MSCintermsofmorphologyandimmunophenotype,theyseemtobesuperiorintermsofproliferation, formingmoreCFUͲFwhenplated(Kernetal.,2006),PLAcells,orAdiposeTissuederivedMSC(ATͲMSC) could be an interesting alternative source for cell therapy and regenerative medicine purpose (H. Mizuno&Hyakusoku,2003)
1.1.1.4.3 Adultstemcellsandtherapeuticapplication
Stem cells are attractive for use in cell based therapies due to the very attribute that define them : because they are selfͲrenewing and can differentiate into mature cell types. In theory stem cells can serveasalimitlesssupplyofcellsandasourceforawiderangeofphenotypes.Inpractice,however, eachtypeofstemcellshasitsownadvantageanddisadvantages.
1.1.2 Placenta,umbilicalcordandfoetalmembranes;roleandphysiology
ofatransitionalorgan
The human placenta is a transitional organ, beginning to develop upon implantation of the blastocyst into the maternal endometrium and is mediator between the mother and fœtus allowing physiologic exchangeprocessesessentialtothedevelopmentoftheembryos.
It is formed of both a maternal and fœtal part, thus present two different genotypes, and is programmed to last for the 9 months of a normal pregnancy, making the fetoͲplacental complex a temporaryallogeneicnaturaltransplantresistanttorejection.
Althoughfœtalandmaternalcirculationareextremelyclosetoeachotherintheplacentatheyremain separated by tissue layer thus forming the placental barrier. While oxygen and nutrients are supplied from the maternal blood to the fœtal circulation, and fœtal blood discharge carbon dioxide and metabolicwasteproducts,makingtheplacentaactduringthepregnancyasthefœtallung,kidneyand intestine in addition to its important role as an endocrine gland. The placenta is thus an autonomous functionalstructure,takingoverimportantregulatoryfunctionsduringpregnancy.
Duringthefirststagesofpregnancytrophoblastcellsareformedontheouterlayeroftheblastocyst, theywillprovidenutrientstotheembryosandultimatelyformalargepartoftheplacenta.Thislayerof trophoblasticcellsarethefirsttoproliferateanddifferentiatefromthefertilizedeggatapproximately6 daysafterfertilizationinhumans.
They will differentiate into two cell layers, the inner layer, cytotrophoblast, single celled, and the syncytiotrophoblast, outer thick multinucleated continuous cell layer that lack cell boundaries, grows into the endometrial stroma and covers the surface of the placenta. It is formed as a result of the differentiationandfusionoftheunderlyingcytotrophoblastcells,aprocessthatcontinuesthroughout placentaldevelopment.ItcontributestothebarrierfunctionoftheplacentaandwillalsosecretehCGin ordertomaintainprogesteronesecretionandsustainpregnancy.
The core of placental villi contain mesenchymal cells and placental blood vessels that are directly connected to the fetal circulation via the umbilical cord. This core is surrounded by two layers of trophoblast; a single layer of mononuclear cytotrophoblast that fuse together to form the overlying multinucleated syncytiotrophoblast layer that covers the entire surface of the placenta. It is this syncytiotrophoblastthatisindirectcontactwiththematernalbloodthatreachestheplacentalsurface, and thus facilitates the exchange of nutrients, wastes and gases between the maternal and fetal systems.
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