—Original Article—
Ultrastructural comparison of porcine putative embryonic stem cells derived by in vitro fertilization and somatic cell nuclear transferHyunju YOO1), Eunhye KIM1), Seon-Ung HWANG1), Junchul David YOON1), Yubyeol JEON1), Kyu-Mi PARK1), Kyu-Jun KIM1), Minghui JIN1), Chang-Kyu LEE2), Eunsong LEE3), Hyunggee KIM4), Gonhyung KIM1) and Sang-Hwan HYUN1)
1)Laboratory of Veterinary Embryology and Biotechnology (VETEMBIO), College of Veterinary Medicine, Chungbuk National University, Cheongju 28644, Republic of Korea
2)Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute for Agriculture and Life Science, Seoul National University, Seoul 08826, Republic of Korea
3)Laboratory of Theriogenology, College of Veterinary Medicine, Kangwon National University, Kangwon 24341, Republic of Korea
4)Department of Biotechnology, School of Life Sciences and Biotechnology, Korea University, Seoul 02841, Republic of Korea
Abstract. Theultrastructureofporcineputativeembryonicstemcellsandporcinefetalfibroblasts(PFFs)wasanalyzedbytransmissionelectronmicroscopy.Theaimofthisstudywastocomparethefeaturesoforganellesinin vitrofertilization(IVF)derivedporcineembryonicstemcells(IVF-pESCs)andsomaticcellnucleartransfer(SCNT)derivedpESCs(SCNT-pESCs).Also,thefeaturesoforganellesinhigh-passageIVF-pESCswerecomparedwiththoseinlow-passagecells.TheultrastructureofPFFsshowedraremicrovillionthecellsurfaces,polygonalorirregularnucleiwithonetotworeticular-shapednucleoliandeuchromatin,lowcytoplasm-to-nucleusratios,rareribosomes,rareroughendoplasmicreticulum,elongatedmitochondria,rich lysosomes and richphagocytic vacuoles. IVF-pESCs showed raremicrovilli on the cell surfaces, roundor irregularnucleiwith one to two reticular-shaped nucleoli and euchromatin, low cytoplasm-to-nucleus ratios, rich ribosomes, longstacksofroughendoplasmicreticulum,elongatedmitochondria,rarelysosomesandrareautophagicvacuoles.Bycontrast,SCNT-pESCsshowedrichmicrovilliwithvariouslengthsandfrequenciesonthecellsurfaces,polygonalnucleiwithonereticular shaped nucleoli and heterochromatin, high cytoplasm-to-nucleus ratios, rare ribosomes, rare rough endoplasmicreticulum,roundmitochondria,richlysosomesandrichphagocyticvacuoleswithclearintercellularjunctions.Furthermore,high-passageIVF-pESCsshowedirregularlyshapedcolonies,pyknosisandnumerouslysosomesassociatedwithautophagicvacuolesshowingsignsofapoptosis.Inconclusion,thisstudyconfirmsthattheultrastructuralcharacteristicsofpESCsdifferdependingontheirorigin.TheseultrastructuralcharacteristicsmightbeusefulinbiomedicalresearchusingpESCs,leadingtonewinsightsregardingregenerativemedicineandtissuerepair.Key words: Embryonicstemcell,Porcine,Transmissionelectronmicroscopy,Ultrastructure
(J. Reprod. Dev. 62: 177–185, 2016)
Embryonicstemcells(ESCs)arederivedfromtheinnercellmass(ICM)ofblastocyst-stageembryos[1–3].ICMcellsareisolated
byimmunosurgeryandculturedonmitomycinC-inactivatedmouseembryonicfibroblasts (MEFs)asfeeder layers[1,2].ESCsareundifferentiated cells that have the capacity for unlimited prolifera-tion and can differentiate into various types of cell or tissue in vivo and in vitro [2,4,5].Pigsareausefulandmeaningfulmodelinmanybranchesofmedicinebecausetheyareimmunologicallyandphysiologicallysimilartohumans[6–8].Itisbelievedthatporcine
ESCs(pESCs)canplayimportantrolesinbiomedicalresearchasmodelsforcelltherapy,regenerativemedicineandtissuerepairinhumans[8–10].Forthesereasons,theestablishmentofapESClinehasbecomeveryimportant.Consequently,manyresearchershaveattemptedtoestablishporcineES,ES-likeorICMcell linesbyusingpreimplantationblastocysts[9,11,12].Furthermore,severalauthorshavereportedestablishmentofpESCsfrompreimplantationblastocysts derived by in vitrofertilization(IVF)andsomaticcellnucleartransfer(SCNT)[13–15].pESCscanproliferatestablyinanundifferentiated state in vitrowithMEFsasfeederlayersandbasicfibroblastgrowthfactor(bFGF)[14–17].SomeofthecharacteristicsofpESCs,includingtheirpluripotency-
relatedmolecularmarkers,karyotypeandsignalingpathways,havebeenreported[14,18].However,detailsof theultrastructureofpESCshavenotbeenreportedpreviously.Transmissionelectronmicroscopy(TEM)isamajoranalysismethodincellbiology[19,20]andausefulmethodincancerresearch,virologyandESCresearch[21–24].TEMtechniquescanprovideusefulinformationaboutthe
Received:September2,2015Accepted:December15,2015PublishedonlineinJ-STAGE:January28,2016©2016bytheSocietyforReproductionandDevelopmentCorrespondence:S-HHyun(e-mail:[email protected])andGKim(e-mail:[email protected])Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionNon-CommercialNoDerivatives(by-nc-nd)License<http://creativecommons.org/licenses/by-nc-nd/4.0/>.
Journal of Reproduction and Development, Vol. 62, No 2, 2016
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functionality of cells. The ultrastructural characteristics of mouse ESCs(mESCs)[25],nonhumanprimateESCs[1]andhumanESCs(hESCs)[26],aswellasembryoidbodies(EBs)derivedfrommESClines[27,28],havebeenreported.Moreover,Talbotet al. reported theultrastructureofporcineblastocysts[29].Porcineblastocystshadnuclei,Golgicomplexes,numerousmitochondria,freeribosomesandpolysomes,verylargelipiddroplets,microfilaments,microtubulesandjunctionalcomplexeswithtightjunctionsanddesmosomes[29].Mostoftheaboveultrastructuralfeaturesweredocumentedby
TEM.However,TEMimagesoftheultrastructureofpESCsderivedbyIVFandSCNThavenotbeenreportedpreviously.Weanalyzedtheultrastructureofporcinefetalfibroblasts (PFFs)andpESCsderivedbyIVFandSCNTbyTEM.TheaimofthisstudywastocomparethefeaturesoforganellesinIVF-pESCsandSCNT-pESCs.SinceitwasrequiredtounderstandtheapoptosisofpESCsduringlong-termculturein vitro,wealsocomparedthefeaturesoforganellesinhigh-passageIVF-pESCswiththoseinlow-passageIVF-pESCs.
Materials and Methods
Ethics statementThisstudywascarriedoutinstrictaccordancewiththerecom-
mendationsintheGuidefortheCareandUseofLaboratoryAnimalsoftheNationalVeterinaryandQuarantineService.TheprotocolwasapprovedbytheCommitteeontheEthicsofAnimalExperimentsofChungbukNationalUniversity(PermitNumber:CBNUA-584-13-01).Allanimalsweresacrificedunderisofluraneanesthesia,andalleffortsweremadetominimizesuffering.
ChemicalsUnlessotherwiseindicated,allchemicalsandreagentsusedin
thepresentstudywerepurchasedfromSigma-Aldrich(St.Louis,MO,USA).
Preparation of the feeder cell layerTheMEFsusedasthefeedercelllayerwerepreparedfromICR
mice.ICRmicewerekilledatpregnancyday13andfetuseswererecovered.Fetalheads,internalorgansandlegswereremoved.Theremainingtissuesweremincedinfreshphosphate-bufferedsaline(PBS)andcentrifugedat2000rpmfor3minatleasttwiceuntilMEFswereobtained.MEFswereculturedinDulbecco’smodifiedEagle’smedium(DMEM,Gibco,Carlsbad,CA,USA)containing10%FBS(Gibco),1%non-essentialaminoacids(Gibco),1%glutamine(Gibco),0.1mMβ-mercaptoethanol(Gibco)and1%antibiotics-antimycotics(Gibco)(growthmedium)at37ºCunder5%CO2inair.MEFswerepassagedtwotothreetimesbeforeinactivationwithmitomycinC(10µg/Ml,Roche,Basel,Switzerland)for2–2.5hforuseincultureofpigblastocysts.InactivatedMEFswereplatedatadensityof5×105cells/mlinafour-welldishcoatedwith0.5%gelatinandcontaininggrowthmedium.TheMEFswereusuallyplated1daybeforeseedingofporcineembryosorICMs.
Cell cultureAllof thepESClineswereestablishedandcharacterizedina
previousstudy[30]. Inbrief,hatchedporcineblastocystswereobtainedbyIVFandSCNTusingin vitromatured(IVM)oocytes.
Oocyte collection and maturation, sperm preparation, donor cell preparation,IVFandSCNTwereperformedaspreviouslyreported[31–33].Theblastocystswerecollected7daysafterIVFandSCNT.ThegrowthmediumofinactivefeedercellswasreplacedwithpESCculturemedium2hbeforeblastocystplating.ThepESCculturemediumconsistedoflow-glucoseDMEM/F10(Gibco)containing1%non-essentialaminoacids,1%glutamine,0.1mMβ-mercaptoethanol,1%antibiotics-antimycotics,4ng/mlbFGF(Invitrogen,Carlsbad,CA,USA)and15%FBS.Blastocystswereremovedfromthezonapellucidausing0.5%protease.Forplating,blastocystswerewashedthreetimesinpESCculturemedium.TheywerethenseededonamonolayerofmitomycinC-inactivatedMEFsinfour-wellplates(Nunc,Roskilde,Denmark).Theplatingefficiencyofprimarycultureswasdeterminedbyscoringthenumberofattachedcoloniesafter48h.Thetimingofthedisaggregationofprimarycolonieswasbasedonmorphologyandsize.Themediumwasreplaceddaily,andnewcoloniesweresubculturedatanintervalofapproximately7–10days,accordingtotheirsizeandgrowthrate.PFFswereisolatedaccordingmethodsinapreviousreport[34]andculturedinDMEM(Gibco)containing10%FBS(Gibco),1%non-essentialaminoacids(Gibco),1%glutamine(Gibco),0.1mMβ-mercaptoethanol(Gibco)and1%antibiotics-antimycotics(Gibco)(growthmedium)at37ºCunder5%CO2inair.TheattachmentandgrowthofPFFswereexamineddaily,andtheculturemediumwasreplacedevery2days.Thecellswereatpassage2.pESClinesderivedbyIVFandSCNTweregrowninmonolayercultureonmitomycinC-treatedMEFs.Seven-day-oldcolonieswere individuallypeeledoff thefeederlayerwithglasscapillariesanddissectedusingtwosyringeneedles.ThepESCmedium,includingbFGF,wasreplacedeveryday.pESCculturewasperformedinmediumat37ºCunder5%CO2 inahumidifiedatmosphere.
Transmission electron microscopyForTEManalysis,twolinesofpESCsderivedfromIVFblastocysts
(IVF0227_P20andIVF0214_P37)andonelineofpESCsderivedfromSCNTblastocysts(TransgenicpESC_P20)linewereprepared.ThedonorcellsusedforSCNTweretransgeniccelllinesoverexpressingthe11betahydroxysteroiddehydrogenase(11β-HSD1)gene.Allof thepESCsweregrownonafeeder layer infour-wellplates.Subsequently,pESCswerepeeledoffthefeederlayerandcollectedintotubes.Furthermore,PFFsweretrypsinizedandcollectedintotubes.Thecellswerewashedwith0.1Mphosphatebuffer(pH7.2)twicefor5min,andcellpelletswereresuspendeddirectlyin2.5%glutaraldehydefixative(EMS,FortWashington,PA,USA)in0.1Mphosphatebuffer(pH7.2)andstoredat4ºCovernight.Then,thecellswerewashedin0.1Mphosphatebuffer(pH7.2)for5minthreetimes.Afterwashing,thecellsweretransferredto1%osmiumtetroxide(OsO4)(EMS)in0.1Mphosphatebuffer(pH7.2)for1handthenwashedfor10minthreetimesinthesamebuffer.Additionally,thecellswerewashedtwiceindistilledwaterfor5mineach.Thefixedcellsweredehydratedinanascendingseriesofethanolsolutions(Merck,Rahway,NJ,USA)(50%,60%,70%,80%,90%,95%and100%;15mineach),andwashedthreetimesin100%ethanolfor15mineach.Thecellswerethenwashedtwicein100%propyleneoxide(EMS)for30mineachandembeddedinEpon812(EMS).Thecellswereinfiltratedwithpropyleneoxide:Epon812(3:1)for
ULTRASTRUCTURALCOMPARISONOFpESCs 179
3hfollowedbypropyleneoxide:Epon812(1:1)overnight.Next,thecellswereinfiltratedwithpropyleneoxide:Epon812(1:3)for3handthen100%Epon812for3h.Subsequentlytheresinwaspolymerizedinanovenat70ºCfor12h.Semithinsectionswerecutwithaglassknife(EMS),allsectionswerepreparedusinganultramicrotome(UltracutUCT,Leica),andultrathinsectionswerecutwithadiamondknife(DiATOME,Hatfield,PA,USA).Semithinsectionswerestainedwithtoluidineblue(EMS).Ultrathinsectionswerefirstcontrastedwith2%uranylacetate(EMS)for10min,rinsedwithdouble-distilledwater,contrastedwithleadcitrate(EMS)for1minandrinsedwithdistilledwater.Finally,thecontrastedultrathinsectionswereexaminedandphotographedundera transmissionelectronmicroscope(JEM-1400Plus,JEOL,Tokyo,Japan).
Results
Ultrastructural analysis of colony shape and microvilliAsshowninTable1andFig.1,thecellsurfaceshowedvarious
shapesofcoloniesandmicrovillidependingon the typeofcellline.PFFsandSCNT-pESCs,asobservedbyTEM,grewasroundorpolygonalcolonies(Fig.1Aand1D).Bycontrast,IVF-pESCsgrewas irregularlyshapedcolonies(Fig.1B).Furthermore, themicrovillihadvariouslengthsandfrequencies(Fig.1A,1Band1D).SCNT-pESCshadnumerousmicrovilli(Fig.1D).Bycontrast,microvillioccurredrarelyonPFFsandIVF-pESCs(Fig.1Aand1B).
Ultrastructural analysis of the nucleusWeanalyzedtheultrastructureofthenucleus,nucleolus,nuclear
envelopeandchromatininallof thecell lines(Table2andFig.2).Thenuclei inPFFshadapolygonalor irregular shapeandcontainedoneortwodensenucleoli(Fig.2Aand2B).ThenucleiinIVF-pESCsweredeeplyinfoldedandofroundorirregularshape(Fig.2C).Moreover,theyhadonetotwoprominentnucleolithatwerereticularshapedanddark(Fig.2C).ThenucleiinSCNT-pESCswerelargeandpolygonal,andtheonenucleoluswasreticularshaped(Fig.2D).AsshowninFig.2,allcelllineshadanuclearenvelope.Moreover,thechromatininthePFFsandIVF-pESCswasobservedaslow-density,electron-lucentnuclearmaterial(Fig.2A,2Band2C).Euchromatinoccupiedmostofthenuclearspace.Conversely,SCNT-pESCshadheterogeneousstructurescontainingeuchromatinandheterochromatin(Fig.2D).Thecytoplasm-to-nucleusratiowaslowinPFFsandIVF-pESCs(Fig.2A,2Band2C)andhighinSCNT-pESCs(Fig.2D).
Ultrastructural analysis of protein-synthesis-associated organellesTheimportant functionofribosomes, theroughendoplasmic
reticulum(rER)andtheGolgiapparatus(Table3andFig.3) isproteinsynthesis.AsshowninTEMmicrographs,therERwasrarelyobservedinPFFsandSCNT-pESCs(Fig.3Aand3D).Furthermore,thecytoplasmofthesecellscontainedfewfreeribosomesandpolysomes.Bycontrast,longstacksofribosome-studdedrERwereobservedinIVF-pESCs(Fig.3Band3C).TherERwasoftenextensiveandrichinfreeribosomes/polysomes.Ontheotherhand,theGolgiapparatuswasrarelyobservedinthecytoplasminallcelllines.
Ultrastructural analysis of intracellular digestion-associated organellesIntracellulardigestion-associatedorganellesincludephagocytic
vacuoles,autophagicvacuolesandlysosomes(Table4andFig.4).RoundphagocyticvacuolescontainingmembranousstructureswerefrequentlyseeninPFFsandSCNT-pESCs,butnotinIVF-pESCs.Autophagicvacuolescontainingdenseirregularbodieswerealsoobserved,althoughtheywerenotseeninPFFsandSCNT-pESCsandwererareinIVF-pESCs.Inaddition,lysosomeswerefrequentlyseeninPFFsandSCNT-pESCsasroundelectron-densecytoplasmicstructures(Fig.4A,4Band4D).Bycontrast,lysosomeswerenotprominentinIVF-pESCs(Fig.4C).
Ultrastructural analysis of mitochondriaMitochondriawithdifferentshapesandsizeswereobservedinall
celllines(Table5andFig.5).Elongatedwell-developedmitochondriawereobservedfrequentlyinPFFs(Fig.5Aand5B).Furthermore,
Table 1. Ultrastructuralcomparisonofcoloniesandmicrovilliinthreecell lines
Origin Cellline
Organelle
Cellsurface
Colonies MicrovilliPFFs Fetalfibroblasts Roundorpolygonal RareIVF IVF0227 Irregular RareSCNT TransgenicpESCs Roundorpolygon Rich
PFFs, porcine fetal fibroblasts; IVF, in vitro fertilization; SCNT,somatic cell nuclear transfer.
Fig. 1. Transmission electron micrographs of the cell surface. (A)Porcine fetal fibroblasts, magnification × 600. (B) In vitro fertilization-derived IVF 0227 pESCs, magnification × 1500.(C) pESC lines co-cultured with mouse embryonic fibroblasts(MEFs), magnification × 3000. n, nucleus; nu, nucleolus; ne,nuclearenvelope;pv,phagocyticvacuole;rer,roughendoplasmicreticulum.(D)TransgenicpESCsderivedbySCNT,magnification×8000.mv,microvilli.
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thecristaeofthePFFsmitochondriaweredistinctandarrangedinparallel.Similarly,elongatedwell-developedmitochondriawereseeninIVF-pESCs(Fig.5C).However,thecristaeofthemitochondriawerenotdistinct.Bycontrast,roundwell-developedmitochondriawereobservedfrequentlyinSCNT-pESCs(Fig.5D).Moreover,thecristaeoftheSCNT-pESCmitochondriaweredistinctandarrangedin parallel.
Ultrastructural analysis of intercellular junctionsIntercellularjunctionsincludingdesmosome-likejunctionsandgap
junctionswereobservedbetweenadjacentcells,andthecellswerecloselyapposed(Table6andFig.6).IntercellularjunctionswereseeninPFFsandSCNT-pESCs,butnotinIVF-pESCs.Desmosome-likejunctionsandgapjunctionswereinfrequentlyobservedinPFFs.Bycontrast,thesejunctionswerefrequentlyseeninSCNT-pESCs.
Ultrastructural comparison of pESCs of various origins with mESCs and hESCsThisisthefirstultrastructuralcomparisonbetweenpESClines
andESCsfromotherspecies[25,26](Table7).Ultrastructuralexaminationshowed that thecolonymorphologyofPFFsand
SCNT-pESCswassimilartothatofmESCs[25].Ingeneral,PFFs,SCNT-pESCsandmESCswereround,whereasIVF-pESCshadanirregularshape.Furthermore,thenucleiofPFFsandSCNT-pESCswerepolygonalandresembledthenucleiofhESCs[26];thenucleiofIVF-pESCsweresimilartothoseofmESCs.PFFs,IVF-pESCs,mESCsandhESCsallshowedeuchromatin,whereasSCNT-pESCs
Table 2. Ultrastructuralcomparisonofthenucleusinthreecelllines
Origin Cellline
Organelle
Nucleus
Nucleus Nucleolus Nuclearenvelope Chromatin C:NratioPFFs Fetalfibroblasts Polygonalorirregular Onetotwo Normal Euchromatin LowIVF IVF0227 Roundorirregular Onetotwo Normal Euchromatin LowSCNT TransgenicpESCs Polygonal One Normal Heterogeneous High
PFFs,porcinefetalfibroblasts;IVF,in vitrofertilization;SCNT,somaticcellnucleartransfer;C,cytoplasm;N,nucleus.
Fig. 2. Transmissionelectronmicrographsofthenucleus.(A,B)Porcinefetalfibroblasts,magnification×5000.(C)In vitrofertilization-derivedIVF0227pESCs,magnification×4000.(D)TransgenicpESCsderivedbySCNT,magnification×10000.n,nucleus;nu,nucleolus;ne,nuclearenvelope.
Table 3. Ultrastructuralcomparisonoftheproteinsynthesis-associatedorganelleinthreecelllines
Origin Cellline
Organelle
Proteinsynthesis
Ribosomes RoughER GolgiapparatusPFFs Fetalfibroblasts Rare Rare RareIVF IVF0227 Rich Rich RareSCNT TransgenicpESCs Rare Rare Rare
PFFs,porcinefetalfibroblasts;IVF,in vitrofertilization;SCNT,somaticcellnucleartransfer;ER,endoplasmicreticulum.
Fig. 3. Transmission electron micrographs of protein-synthesis-associatedorganelles.(A)Porcinefetalfibroblasts,magnification× 5000. (B) In vitro fertilization-derived IVF 0227 pESCs,magnification × 4000. (C) In vitro fertilization-derived IVF0227 pESCs, magnification × 6000. (D) Transgenic pESCsderivedbySCNT,magnification×10000.r,ribosome;rer,roughendoplasmicreticulum;g,Golgiapparatus.
ULTRASTRUCTURALCOMPARISONOFpESCs 181
showedheterogeneousstructurescontainingheterochromatinandeuchromatin.Additionally,thecytoplasm-to-nucleusratiowaslowinPFFs,IVF-pESCsandmESCsandhigh inSCNT-pESCsandhESCs.Allmitochondria inPFFs,IVF-pESCsandhESCswerewelldevelopedandelongated.Bycontrast,well-developedroundmitochondriawereobservedinSCNT-pESCsandmESCs.
Ultrastructural analysis of high-passage IVF-pESCsAsshowninTable8andFig.7,TEMexaminationshowedthatthe
organellesofhigh-passageIVF-pESCs(IVF0214)weredifferentfromthoseoflow-passageIVF-pESCs(IVF0227).Pyknosisandwrinklednuclearenvelopeswereevidentinhigh-passageIVF-pESCs,butnotinlow-passageIVF-pESCs(Fig.2Cand7A,7C,7F).Furthermore,thechromatininlow-passageIVF-pESCswaslow-densityeuchro-matin,whereashigh-passageIVF-pESCsdisplayedheterogeneousstructurescontainingheterochromatinandeuchromatin(Fig.2Cand7C).Additionally,autophagicvacuolesandlysosomeswerefrequentlyobservedinhigh-passageIVF-pESCsbutwereinfrequentinlow-passageIVF-pESCs(Fig.4Cand7D,7F).TEMexaminationfurthershowedthattheorganellesofhigh-passageIVF-pESCsweresimilar to thoseofdifferentiatedhESCs(Table9).Thenuclei inhigh-passageIVF-pESCsanddifferentiatedhESCsshowedpyknosisandwrinklednuclearenvelopes(Fig.7C).Moreover,high-passageIVF-pESCsanddifferentiatedhESCshadahighchromatindensity.Additionally,therERandlysosomeswerefrequentlyobservedinhigh-passageIVF-pESCsanddifferentiatedhESCs(Fig.7Aand7B).
Table 4. Ultrastructuralcomparisonoftheintracellulardigestion-associatedorganelleinthreecell lines
Origin Cellline
Organelle
Intracellulardigestion
Phagocyticvacuole Autophagicvacuole LysosomePFFs Fetalfibroblasts Rich Absent RichIVF IVF0227 Absent Rare RareSCNT TransgenicpESCs Rich Absent Rich
PFFs,porcinefetalfibroblasts;IVF,in vitrofertilization;SCNT,somaticcellnucleartransfer.
Fig. 4. Transmission electron micrographs of intracellular digestion-associated organelles. (A, B) Porcine fetal fibroblasts,magnification×5000.(C)In vitrofertilization-derivedIVF0227pESCs, magnification × 4000. (D) Transgenic pESCs derivedbySCNT,magnification×10000. pv, phagocytic vacuole; apv,autophagicvacuole;ly,lysosome.
Fig. 5. Transmission electron micrographs of mitochondria. (A)Porcinefetalfibroblasts,magnification×5000.(B)Porcinefetalfibroblasts,magnification×6000.(C)In vitrofertilization-derivedIVF0227pESCs,magnification×6000. (D)TransgenicpESCsderivedbySCNT,magnification×10000.m,mitochondrion.
Table 5. Ultrastructuralcomparisonofthemitochondrioninthreecelllines
Origin Cellline
Organelle
Mitochondrion
Development ShapePFFs Fetalfibroblasts Well ElongatedIVF IVF0227 Well ElongatedSCNT TransgenicpESCs Well Round
PFFs, porcine fetal fibroblasts; IVF, in vitro fertilization; SCNT,somatic cell nuclear transfer.
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Discussion
ThisstudywasthefirsttocomparetheultrastructureofdifferentpESClinesusingTEM.Weobservedmicrovilli,nucleicontainingreticulatednucleoli,rERs,Golgiapparatuses,lysosomesandmito-chondriainthepESClinesderivedfromvariousorigins.ComparedwithPFFsandIVF-pESCs,SCNT-pESCshadmoremicrovilliontheirsurfaces,whichsuggeststhattheyhavehighabsorptionand
secretoryactivity, resulting inan increase incell surfacearea.Microvilli indicatehighlymetabolicactivityandhavealsobeenobservedinmESCs,mouseEBsandhESCs[25–27].Largeorsmalldeeplyinfoldedeuchromatin-orheterochromatin-
containingnucleiwithonetothreereticular-shapednucleoliandanuclearenvelopeweregenerallyseeninallpESClines.ThesefeaturesindicatethatthenucleuscontrolsgeneexpressionandmediatesDNAreplicationduringthecellcycle.TheTEMappearanceofthenucleuswassimilartothatreportedinotherhESCsandmESCs[25,26].Also,thenuclearshapeandstructureweresimilartothoseintheblastocystsofmanymammalianspecies[35–37].NucleoliweretheprominentcontrastedstructuresinthenucleiofallpESClinesobservedbyTEM.Inmostcells,thenucleuscontainedoneorafewnucleoli.Reportedly,mammaliannucleicontainoneorafewnucleoli,andthesizeandorganizationofthenucleoliaredirectlyrelatedtoribosomeproduction[38,39].Furthermore,weobservedheterochromatininSCNT-pESCsandhigh-passageIVF-pESCs.Thisresult indicatesthatthechromatininSCNT-pESCsandhigh-passageIVF-pESCsishighlycondensedandistypicallynottranscribed[40,41].InPFFsandIVF-pESCs,butnotSCNT-pESCs,thenucleus-to-cytoplasmratiowaslow,whichmayindicatehighmaturityofSCNT-pESCs.Inthisstudy,therERwasseentobeincontactwithribosomes.
Similarobservationshavebeenreportedinmammalianembryos,blastocystsandcells[35,37,42,43].Also,therERhasbeendescribedinporcineblastocysts[29].Additionally, thepatternsofrERand
Fig. 6. Transmissionelectronmicrographsofintercellularjunctions.(A)Porcine fetal fibroblasts, magnification × 1500. (B)TransgenicpESCsderivedbySCNT,magnification×10000.gj,gapjunction;dj,desmosome-likejunction.
Table 7. UltrastructuralcomparisonofpESCsofvariousoriginswithmESCsandhESCs
OrganelleOrigin OtherspeciesofESCs
PFFs IVF SCNT Mouse (Baharvandet al.2003)
Human (Sathananthanet al.2002)
Colony Roundorpolygonal Irregular Roundorpolygonal Round SaucerNucleus Polygonalorirregular Roundorirregular Polygonal Roundorirregular PolygonalNucleolus Onetotwo Onetotwo One One to three One to three Chromatin Euchromatin Euchromatin Heterogeneous Euchromatin EuchromatinC:Nratio Low Low High Low HighMitochondrion Elongated Elongated Round Round Elongated
PFFs,porcinefetalfibroblasts;IVF,in vitrofertilization;SCNT,somaticcellnucleartransfer;C,cytoplasm;N,nucleus.
Table 8. Ultrastructuralcomparisonoflow-andhigh-passageinpESCsderivedbyIVF
Organelle
Cellline
In vitrofertilizationderived
IVF0227 IVF0214
Lowpassage(20th) Highpassage(37th)Colony Irregular IrregularNucleus Roundorirregular PyknosisandirregularNuclearenvelope Normal WrinkleChromatin Euchromatin HeterogeneousAutophagicvacuole Rare RichLysosome Rare RichMitochondrion Elongated Poorandelongated
Table 6. Ultrastructuralcomparisonof intercellular junctions inthree cell lines
Origin Cellline
Organelle
Intercellularjunctions
Desmosome-like GapPFFs Fetalfibroblasts Rare RareIVF IVF0227 Absent AbsentSCNT TransgenicpESCs Rich Rich
PFFs,porcinefetalfibroblasts;IVF,in vitrofertilization;SCNT,somatic cell nuclear transfer.
ULTRASTRUCTURALCOMPARISONOFpESCs 183
ribosomefrequencyfoundhereweresimilar tothosereportedinhESCsandmESCs[25,26].AsshowninTEMmicrographs,therERisformedinallpESClinesbyseriesofstacksarrangedinparallel.Ontheotherhand,theGolgiapparatuswasrarelyobservedandhadflattenedcisternaeinallpESClines,possiblyindicatinglowactivityofthisorganelle.Thislowactivitycouldleadtodecreasedproteinsecretion.ThisfindingmayreflecttheproteinquantitiesrequiredbyallpESClinestoproliferate.TheGolgiapparatusisinvolvedinproteinsynthesisandexportofcellularproductsforsecretion[44–46].TheGolgiapparatushasalsobeendescribedinporcineepiblastcells[29].Phagocyticvacuolesandlysosomesperformacentral role in
intracellulardigestion.TheywereobservedfrequentlyinPFFsandSCNT-pESCsbutwererare inIVF-pESCs.Phagocyticvacuolesand lysosomeswereobserved in theblastocystsofmammalianspecies[37,47,48]andinhumanandbovineembryos[43,47].Thelysosomeisacellularorganellethatcontainsacidhydrolasestodegradedeliveredmaterials.Digestionofphagocyticvacuolesbytheenzymescontainedwithinlysosomesreleasestheirnutrientsintothecytoplasm[49].EvidenceofphagocytosiswasobservedinPFFsandSCNT-pESCs.Phagocytosisisinvolvedintheacquisitionofnutrientsbycells.Furthermore,itiscriticalfortheuptakeanddegradationofinfectiousagentsandsenescentcellsandcontributestodevelopment,tissueremodeling, the immuneresponseandinflammation[50].AutophagicvacuoleswerenotobservedfrequentlyinthecytoplasmofanypESCline.However,thepresenceofautophagicvacuolesinhigh-passage IVF-pESCssuggests thatautophagyhas rolesincatabolism,degradationandproductionofaminoacidsunderstarvationconditions,recyclingofcellularcomponents,preventionofvariousdiseasesandcelldeath[51,52].Thesefindingssuggestthatautophagyisanadaptiveresponsetostressthatpromotessurvival,whereasinothercases,itappearstopromotecellmorbidity.In thepresent study,mitochondria inallpESC linesvaried
insizeandshapeandhadmostly tubularcristae.ElongatedandroundmitochondriaweredetectedinallpESClines.Theelongatedmitochondria resembled those found in porcine blastocysts and hESCs[26,29].Bycontrast, roundmitochondriaarefrequentlyfoundinmESCs[25].Furthermore,wefoundthatwell-developedmitochondriawerepresentatahighfrequencyinall threepESClines.Previousreportsdemonstratedthatadultbovineoocyteshave
Fig. 7. Transmission electron micrographs of in vitro fertilization-derivedIVF0214pESCs(highpassageandculturedin vitro after 37passages).(A)Ultrastructureofanucleus-associatedorganelle,magnification×1500. (B)Ultrastructureofaprotein-synthesis-associated organelle, magnification × 2000. (C) Ultrastructureof a nucleus-associated organelle, magnification × 3000. (D)Ultrastructure of an intracellular digestion-associatedorganelle,magnification × 3000. (E) Ultrastructure of a mitochondrion-associated organelle, magnification × 5000. (F) Ultrastructureof a nucleus-associated organelle, magnification × 5000. n,nucleus;nu,nucleolus;ne,nuclearenvelope;m,mitochondrion;pv,phagocyticvacuole;apv,autophagicvacuole;ly,lysosome;v,vesicle;ld,lipiddroplets;rer,roughendoplasmicreticulum;mv,microvilli.
Table 9. UltrastructuralcomparisonofpESCs(IVF-highpassage)withmESCsandhESCs
Organelle
Cellline Other species
PorcineESCs(IVF0214) MouseESCs (Baharvandet al.)
HumanESCs (Sathananthanet al.)
Highpassage Undifferentiated Undifferentiated DifferentiatedColony Irregular Round Saucer GobletNucleus Pyknosisandirregular Roundorirregular Polygonal PyknosisNuclearenvelope Wrinkle Normal Normal WrinkleChromatindensity High Low Low HighRoughER Rich Rich Rare RichLysosome Rich Rare Rare RichMitochondrion Poorandelongated Round Elongated Elongated
C,cytoplasm;N,nucleus;ER,endoplasmicreticulum.
YOO et al.184
a largermitochondrialpopulationcomparedwithcalfoocytes,suggestingthattheadultbovineoocytesaremature[53].Moreover,themitochondrialocalizationofstriatedductswasalsoclear.PFFsandIVF-pESCscontainelongatedmitochondriawithadensematrix,whereasSCNT-pESCscontain roundmitochondriawithapalematrix.ThesefindingssuggestthatthedifferencesinmitochondrialstructureamongpESClineswereaccompaniedbya functionaldifference[54].Furthermore, thepresenceofmitochondriaisanindicationofmetabolicactivity[55].Also,severalstudiesshowedthatmitochondriaareinvolvedinotherprocesses,suchassignaling,ATPproduction,energymetabolism,cellulardifferentiationandcelldeath,aswellascontrolofthecellcycleandcellgrowth[54,56,57].Mitochondrialcristaearefoldsofthemitochondrialinnermembranethatprovideanincreaseinsurfacearea[56].Thestudyofmitochondrialfunctionhasbecomecentraltoawidevarietyofclinicalandbasicscienceresearch[58,59].ContactareasofsimilarappearancewereobservedinPFFsand
SCNT-pESCs,inwhichtheywereoftenassociatedwithgapjunctionsanddesmosome-likejunctions.Thesejunctionsarethoughttoholdcellstogetherandfacilitatecommunicationbetweenneighboringcells[60,61].Previousreportsdemonstratedgapjunctionsanddesmosome-likejunctionsinmanymammalianembryosandblastocysts[29,35,37,42,47,62–64].However,gapjunctionsanddesmosome-likejunctionswerenotobservedinhESCsandmESCs[25,26].TEManalysisofhigh-passage IVF-pESCsrevealedsignsof
apoptosis(Fig.7).Apoptosisisastrictlyregulatedmechanismfortheorderedremovalofagedordamagedcells[65,66].Ingeneral,ultrastructuralanalysesoflow-passageIVF-pESCsshowednormalnuclei, fewlysosomes, fewautophagicvacuolesandelongatedmitochondria.Bycontrast,high-passageIVF-pESCshadpyknosis,wrinklynuclearenvelopes,numerouslysosomesassociatedwithautophagicvacuolesandpoormitochondria.Featuresofapoptosisincludechromatincondensation,nuclearfragmentation,apoptoticbodiesandlackofmitochondrialswelling[65–69].Therefore,thehigh-passageIVF-pESCsshowedtheinitialsignsofapoptosis.ThisstudyconfirmsthatpESCsofdifferentoriginshaveachar-
acteristicultrastructure.Weidentifieddifferencesandsimilaritiesamong thepESClines.The resultsof thisstudyshowthat theultrastructuralfeaturesofPFFsaresimilartothoseofSCNT-pESCs,butnotIVF-pESCs.Thispresumablyindicatesthatthecellshavedifferentstates.Furthermore,thisstudydemonstratedthatthefeaturesoforganellesareorigindependent.Previousreportsdemonstratedtheultrastructureofporcineembryosandblastocysts[29,37,70,71].Also,TEMpermitsprecisedemonstrationoftheultrastructureofvariousESCs[25,26].However, thepresentstudyis thefirstreportoftheultrastructureofpESCs.Inconclusion,thisstudyconfirmedthattheultrastructuralcharac-
teristicsofpESCsdifferdependingontheirorigin.ThecomparisonofthedifferentpESClinesprovidesusefulinformationregardingtheultrastructureofpESCs.TheultrastructuralcharacteristicsmightfacilitatebiomedicalandhistologicalresearchonpESCs.Also,theultrastructureofpESCscouldplayanimportantroleincelltherapy,regenerativemedicine,tissuerepairanduseofpESCsasahumancellbiologymodel.
Acknowledgments
Thisworkwas supported, inpart, by the intramural researchgrantofChungbukNationalUniversityin2015,andagrantfromthe Cooperative Research Program for Agriculture Science &TechnologyDevelopment (ProjectNo.PJ011077,PJ011288),Ru-ralDevelopmentAdministration,andNationalResearchFounda-tionofKoreaGrants fundedby theKoreanGovernment (NRF-2013R1A2A2A04008751),RepublicofKorea.
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