—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:shhyun@cbu.ac.kr)andGKim(e-mail:ghkim@cbu.ac.kr)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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