Human Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer

18 Pages·2013·4.34 MB·English

by  Masahito Tachibana

Pleasecitethisarticleinpressas:Tachibanaetal.,HumanEmbryonicStemCellsDerivedbySomaticCellNuclearTransfer,Cell(2013),
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Human Embryonic Stem Cells Derived
by Somatic Cell Nuclear Transfer
MasahitoTachibana,1PaulaAmato,2MichelleSparman,1NuriaMartiGutierrez,1RebeccaTippner-Hedges,1HongMa,1
EunjuKang,1AlimujiangFulati,1Hyo-SangLee,1,6HathaitipSritanaudomchai,3KeithMasterson,2JanineLarson,2
DeborahEaton,2KarenSadler-Fredd,2DavidBattaglia,2DavidLee,2DianaWu,2JeffreyJensen,1,4PhillipPatton,2
SumitaGokhale,5RichardL.Stouffer,1,2DonWolf,1andShoukhratMitalipov1,2,*
1DivisionofReproductive&DevelopmentalSciences,OregonNationalPrimateResearchCenter,OregonHealth&ScienceUniversity,505
NW185thAvenue,Beaverton,OR97006,USA
2DivisionofReproductiveEndocrinology,DepartmentofObstetricsandGynecology,OregonHealth&ScienceUniversity,3181SWSam
JacksonParkRoad,Portland,OR97239,USA
3DepartmentofOralBiology,FacultyofDentistry,MahidolUniversity,Bangkok10400,Thailand
4Women’sHealthResearchUnit,OregonHealth&ScienceUniversity,3303SWBondAvenue,Portland,OR79239,USA
5BostonUniversitySchoolofMedicine,72EastConcordStreet,Boston,MA02118,USA
6Presentaddress:LaboratoryAnimalCenter,OsongMedicalInnovationFoundation,Chungbuk363-951,RepublicofKorea
*Correspondence:[email protected]
http://dx.doi.org/10.1016/j.cell.2013.05.006
SUMMARY cloning, the nature of reprogramming oocyte factors and their
mechanismofactionremainlargelyunknown.
Reprogramming somatic cells into pluripotent em- Inhumans,SCNT wasenvisioned asa meansofgenerating
bryonic stem cells (ESCs) by somatic cell nuclear personalizedembryonicstemcellsfrompatients’somaticcells,
transfer(SCNT)hasbeenenvisionedasanapproach which could be used to study disease mechanisms and ulti-
forgeneratingpatient-matchednucleartransfer(NT)- matelyforcell-basedtherapies(Lanzaetal.,1999;Yangetal.,
ESCs for studies of disease mechanisms and for 2007). However, the derivation of human nuclear transfer-em-
bryonic stem cells (NT-ESCs) has not been achieved despite
developingspecifictherapies.Pastattemptstopro-
numerous attempts during the past decade. The roadblock
ducehumanNT-ESCshavefailedsecondarytoearly
responsible for failure is early embryonic arrest of human
embryonicarrestofSCNTembryos.Here,weidenti-
SCNTembryosprecludingderivationofstableNT-ESCs.Typi-
fied premature exit from meiosis in human oocytes
cally,SCNTembryosfailtoprogressbeyondtheeight-cellstage,
and suboptimal activation as key factors that are
presumably due to an inability to activate critical embryonic
responsible for these outcomes. Optimized SCNT genesfromthesomaticdonorcellnucleus(Eglietal.,2011;Nog-
approaches designed to circumvent these limita- gleetal.,2011).Inafewcases,whenSCNTembryosdidreach
tions allowed derivation of human NT-ESCs. When theblastocyststage,eitherstableESCswerenotrecoveredor
applied to premium quality human oocytes, NT- derivation was not attempted (Fan et al., 2011; French et al.,
ESClineswerederivedfromasfewastwooocytes. 2008). Though the underlying cause of early developmental
NT-ESCs displayed normal diploid karyotypes and arrest remains unclear, mostof thesestudies involving human
oocytesappliedSCNTprotocolsdevelopedfornonprimatespe-
inherited their nuclear genome exclusively from
cies.Previously,wedemonstratedthatSCNTprocedures,when
parental somatic cells. Gene expression and differ-
adaptedtoprimates,succeededinreprogrammingrhesusma-
entiation profiles in human NT-ESCs were similar to
caque adult skin fibroblasts into NT-ESCs (Byrne et al., 2007;
embryo-derived ESCs, suggesting efficient reprog-
Sparman et al., 2009). Therefore, we reasoned that, similar to
rammingofsomaticcellstoapluripotentstate.
other mammals, human MII oocytes must contain reprogram-
mingactivity.
INTRODUCTION Severalrecentobservationsarerelevant.Removalofhuman
oocytes’nucleargeneticmaterial(chromosomes)negativelyim-
Cytoplasmic factors present in mature, metaphase II (MII)-ar- pacts the cytoplast’s subsequent ability to induce reprogram-
restedoocyteshaveauniqueabilitytoresettheidentityoftrans- ming(Noggleetal.,2011).However,whenasomaticcellnucleus
planted somatic cell nuclei to the embryonic state. Since the istransplantedintoanintactoocytecontainingitsownchromo-
initialdiscoveryinamphibians(Gurdon,1962),somaticcellnu- somes,theresultingpolyploid embryosareableto developto
cleartransfer(SCNT)successinarangeofdifferentmammalian blastocystsandsupportESCderivation.Onepossibleexplana-
specieshasdemonstratedthatsuchreprogrammingactivityin tionfortheseobservationsisthatcriticalreprogrammingfactors
enucleated or spindle-free oocytes (cytoplasts) is universal inhumanMIIoocytesarephysicallyassociatedwiththechromo-
(Campbelletal.,1996;Solter,2000;Wilmutetal.,1997,2002). somesorspindleapparatusandaredepletedorcriticallydimin-
However, despite numerous applications of SCNT for animal ishedupon enucleation.Alternatively,itispossiblethatoneor
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Figure 1. Development of Monkey SCNT
Embryos Reconstructed with Optimized
Protocols
AlthoughHVJ-Efusionwasefficient,SCNTcon-
structsrequiredactivationbyelectroporationfor
blastocyst formation. Elimination of ionomycin
from the activation treatment further improved
blastocyst development. I, ionomycin; DMAP,
6-DMAP;CM,compactmorula.
SeealsoTablesS1andS2.
virus of Japan (HVJ-E) to fuse nuclear
donor cells with enucleated MII oocytes
while maintaining cytoplasts in meiosis
(Tachibana et al., 2009). Because of
limitedoocyteavailability,wefirsttested
both the feasibility and efficacy of HVJ-
moreofthestepsinSCNT—namely,oocyteenucleation,donor E-based cell fusion on the development of rhesus macaque
cell nucleus introduction, or cytoplast activation—negatively SCNTembryos.
impactcytoplastquality,renderingitincapableofinducingsuffi- Thefusionrateofadultfibroblastswithcytoplastswas100%
cientreprogramming. after HVJ-E treatment; however, and unexpectedly, the SCNT
In considering distinct biological features of human oocytes embryosgeneratedbyHVJ-Efusionfailedtoprogressbeyond
that could be involved, we focused on our recent observation thecompactmorula(CM)stagefollowingstandardionomycin/
that meiotic arrest in human MII oocytes is unstable and can DMAP (I/DMAP) activation. We previously demonstrated that
be easily perturbed by mechanical manipulations (Tachibana monkey SCNT embryos produced by electrofusion developed
etal.,2013).Earlier,wesuggestedthatretentionofmeiosis-spe- into blastocysts (Byrne et al., 2007; Sparman et al., 2009).
cificactivitiesinthecytoplastiscriticalfornuclearremodelingaf- Therefore, we postulated that exposure of the cytoplast to an
tertransplantationofaninterphase-stagesomaticcellnucleus electropulse (electroporation) could bebeneficial for SCNT re-
(Mitalipovetal.,2007).Thisremodelingispositivelycorrelated programming, perhaps as a supplemental activation stimulus.
with onward development of SCNT embryos after activation. Totestthispossibility,weexposedHVJ-E-fusedSCNTembryos
Therefore,wesystematicallyevaluatedmodificationsinoocyte toelectroporationbeforethestandardI/DMAPactivationtreat-
enucleationanddonorcellintroductionthatmightworktoretain ment.TenpercentofSCNTembryoswerecapableofreaching
meiosis factors in human cytoplasts. We also determined that theblastocyststage(Figure1).Interestingly,thisSCNTblasto-
routinecytoplastactivationtreatmentswereinsufficienttosup- cyst formation rate was unaffected even when exposure to
portsubsequenthumanSCNTembryodevelopment.Weinitially ionomycin was omitted and SCNT embryos were activated
usedrhesusmacaqueoocytestoevaluatefactorsaffectingsuc- with electroporation followed by DMAP treatment (Figure 1).
cessful SCNT reprogramming in a primate system. Subse- Together,theseresultsindicatethat,althoughanelectroporation
quently,werefinedSCNTapproacheswithhigh-qualityhuman stimulusisnotrequiredforcellfusion,itissupportiveofproper
oocytesdonatedbyhealthyvolunteersanddemonstratedthat cytoplastactivationfollowingSCNT.
methodologicalalterationssignificantlyimproveblastocystfor- Histone deacetylase inhibitors, such as trichostatin A (TSA),
mation from human SCNT embryos. Moreover, we derived have been associated with improved SCNT reprogramming in
severalhumanNT-ESClinesfromtheseembryosandvalidated severalmammalianspecies(Dingetal.,2008;Kishigamietal.,
thattheirnuclearDNAisanexclusivematchtoparentalsomatic 2006; Li et al., 2008). We previously demonstrated enhanced
cells,whereasmitochondrialDNAoriginatedalmostexclusively development of monkey SCNT embryos treated with 37.5 nM
fromoocytes.Wealsoconductedextensivepluripotencyassays TSA(from4%upto18%blastocystdevelopmentrate[Sparman
onhumanNT-ESCstoverifyreprogramming. etal.,2010]).However,blastocystqualityandpotentialtogive
risetostableESCsremainedunknown.Here,weplated16mon-
RESULTS keySCNTblastocystsproducedduringTSAtreatmentonmitot-
icallyinactivatedmouseembryonicfibroblast(mEF)feeders,but
SCNTProtocolOptimizationinaNonhumanPrimate noneresultedinNT-ESClineisolation(TableS1).Wereasoned
Model that,althoughTSAtreatmentwaspromotingblastocystforma-
OurrecentstudiesdemonstratedhumanMIIoocytesensitivityto tion,highTSAconcentrationsmaynegativelyaffectblastocyst
prematureactivationinducedbyremovalandreintroductionof quality and epiblast lineage integrity. Therefore, we tested
meioticspindles(Tachibanaetal.,2013)andtotheuseofelec- severallowerTSAconcentrations,aswellasshorterexposure
trofusioninthecontextofcytoplastactivation(Tachibanaetal., times,onmonkeySCNTblastocystdevelopmentandESCisola-
2009).Consequently,ourpresentinvestigationbeganwithopti- tion.ReducingtheTSAconcentrationto10nMorshorteningthe
mizing the use of envelope from inactivated hemagglutinating TSA exposure time from 24 to 12 hr did not affect blastocyst
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Figure2. SCNTBlastocystDevelopmentIs
AffectedbyPrematureCytoplastActivation
(A)Morphologyofnucleardonorfetalfibroblasts
before SCNT. (B) Poor-quality human SCNT
blastocyst without distinct ICM produced with
suboptimal protocols; note the presence of
excludedcells.
(C)Spindle-likestructuresdetectedwhendonor
nucleiwereintroducedintointactMIIoocytes,but
not when introduction was conducted after
enucleation. Arrowhead and arrow point at the
maternal MII spindle and somatic cell spindle,
respectively.
(D)Somaticnuclearcellspindleswereformedin
cytoplasts when oocyte enucleation and fusion
wereconductedinthepresenceofcaffeine.
(E)HumanSCNTblastocystwithprominentICM
(asterisk)producedaftercaffeinetreatment.
(F)NT-ESCcolonywithtypicalmorphologyderived
fromacaffeine-treatedSCNThumanblastocyst.
dard ovarian stimulation protocols and
transvaginalfollicularaspirations.Human
dermal fibroblasts of fetal origin (HDF-f)
synchronized in G0/G1 cell-cycle phase
wereusedasnucleardonors(Figure2A).
Spindle removal and HVJ-E-assisted
donorcellfusionwerecarriedoutwithin
60minofoocyteretrieval.
Most oocytes (95.2% [60 out of 63])
survived MII spindle removal conducted
under polarized microscopy (Oosight)
(Byrne et al., 2007; Sparman et al.,
2009), and nuclear donor fibroblasts
were introduced with100% efficacy us-
ing HVJ-E based fusion. Somatic cell
nucleididnotformspindle-likestructures
thatweredetectable bynoninvasiveex-
amination under polarized microscopy.
Immediatelyafterconfirmationoffusion,
oocyteswereactivatedwithelectropora-
tion/DMAP(4hr)andexposedto10nM
TSAfor12hr.Mostembryos(81.7%[49
out of 60]; Figure 3A, without caffeine
group) formed one or two pronuclei at
the time of removal from TSA, whereas
a slightly higher portion of embryos
cleaved(86.7%[52outof60]),suggest-
rates(TableS1).However,onlySCNTblastocystsproducedwith ing that some SCNT embryos did not exhibit visible pronuclei
10nMTSAsupportedderivationofstablemonkeyNT-ESClines, at the time of examination (Figure 3A). Most cleaved embryos
thoughthenumberofplatedblastocystswassmall(TableS2). developed to the eight-cell stage (61.5% [32 out of 52]), but
Weconcludedthattheseoptimizedprotocolsinanonhumanpri- few progressed to compact morula (13.5% [7 out of 52]) and
matemodelwereadequatetoserveasastartingpointforfurther blastocyst (11.5% [6 out of 52]) stages (Figure 3A). Activation
testingwithhumanoocytes. ofembryonicgenesandtranscriptionfromthetransplantedso-
matic cell nucleus are required for development of SCNT em-
ProducingHumanSCNTBlastocystsandNT-ESCLines bryos beyond the eight-cell stage (Egli et al., 2011; Noggle
Initially, human MII oocytes from healthy volunteers were et al., 2011). Therefore, these results are consistent with the
exposedtotheSCNTapproachthatproducedthebestresults premisethatourmodifiedSCNTprotocolsupportsreprogram-
inthenonhumanprimate.Oocyteswereretrievedfollowingstan- mingofhumansomaticcellstotheembryonicstate.
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duringspindleremovalbutdeclineduetospontaneousactiva-
tion,wefocusedonnoninvasivetreatmentstomaintainmeiotic
arrestduringmanipulation.
Wereportedpreviouslythattheexposureofmonkeyoocytes
to caffeine, a protein phosphatase inhibitor, was effective in
protectingthecytoplastfromprematureactivationandimproved
development of SCNT embryos (Mitalipov etal.,2007). There-
fore,humanoocytesweremaintainedin1.25mMcaffeineduring
spindleremovalandsomaticcellfusion.Asexpected,somatic
cell nuclei introduced into cytoplasts under these conditions
efficiently formed spindle-like structures that were detectable
under birefringence microscopy (83.3% [10 out of 12]) (Fig-
ure 2D). More importantly, the blastocyst development rate of
caffeine-treated embryos was notably enhanced (23.5%)
comparedtothestandardSCNTgroup(Figure3A,withcaffeine
group),andblastocystswerecharacterizedbyvisibleandprom-
inentICMs,similartothoseobservedforIVF-producedembryos
(Figure2E).
Remarkably, when eight SCNT blastocysts produced with
caffeineexposurewereutilizedforESCsisolation,allattached
to mEFs and four formed ICM outgrowths (Figure 3B), which
gaverisetoESC-likecoloniesuponmanualsplittingontofresh
mEF plates (Figure 2F). Subsequent passaging resulted in the
propagation of stable ESC colonies with typical morphology
andgrowthcharacteristics.ThissurprisinglyhighESCderivation
ratewassimilartothatreportedinourpreviousstudywithhuman
IVF-derived blastocysts (50%) and was even higher than in
Figure 3. Development of Human SCNT Embryos and NT-ESC
manipulatedspindletransferembryos(38%)(Figure3B)(Tachi-
DerivationafterCaffeineTreatment
banaetal.,2013).
(A)ImprovedblastocystdevelopmentofhumanSCNTembryostreatedwith
Collectively,ourfindingsindicatethataprotocoldevelopedin
caffeine.Atotalof63(fivecycles)and43(threecycles)oocyteswereutilized
forSCNTwithoutorwithcaffeine,respectively.Sixty(95.2%)and42(97.7%) the monkey model supported blastocyst development for hu-
oocytessurvivedafterSCNTmicromanipulations. man SCNT embryos. However, poor SCNT blastocyst quality
(B)NT-ESCswerederivedonlyfromblastocystsproducedwithcaffeine. precludedESCisolation.Subsequentincorporation ofcaffeine
SeealsoFiguresS1,S2andS3. during enucleation and fusion allowed improved blastocyst
developmentandESClinederivation.
Ofnote,humanSCNTblastocystsexhibitedpoorlyorganized ReproducibilityofHumanSCNTResults
trophectoderm and small or undetectable inner cell masses Interestingly,allfourhumanNT-ESClineswerederivedfromoo-
(ICMs). In addition, large blastomere-like cells were often cytesretrievedfromoneeggdonor(eggdonorA).Eightmature
excluded(Figure2B).Nevertheless,weplatedsixSCNTblasto- MIIoocyteswererecoveredafterasinglestimulationcycle.Us-
cystsontofeederlayerstoexaminetheirabilitytosupportESC ingfetaldermalfibroblastsasnucleardonorcellsandfollowing
derivation. Though four blastocysts attached to mEFs, only our caffeine-incorporated SCNT protocol, five blastocysts
one gave an outgrowth that, after further passaging, failed to were produced (62.5%) that gave rise to four NT-ESC lines
producestableESC-likecells(Figure3B). (80%)(FigureS1availableonline).Inthecontextofgenerating
ThoughtheobservedSCNTblastocystdevelopmentratewas patient-specific pluripotent stem cells, reproducible results
encouraging,furtheroptimizationofthehumanprotocolfocused with various patient-derived somatic cells and with different
onimprovementsinembryoquality.Toassesswhetherspindle eggdonorsareanecessity.
removalinhumanoocytescouldcausespontaneousexitfrom Wethereforeacquiredaskinfibroblastculturefromapatient
meiosis, we introduced somatic nuclei into intact MII oocytes with Leigh syndrome. A total of 15 and 5 MII oocytes were
andexaminedtheirbirefringencepropertiesunderpolarizedmi- collected from two unrelated egg donor volunteers (B and C)
croscopy. All introduced somatic cell nuclei efficiently formed andwereusedforSCNTwiththesefibroblasts.Alloocytessur-
spindle-likestructuresthatwerevisiblewithin30minoffusion vivedspindleremovalandsuccessfullyfusedwithnucleardonor
(17 out of 17) (Figure 2C). Again, spindle formation was not cells.Followingactivationandculture,four(27%,[4outof15])
observedwhensomaticcellnucleiwerefusedwithmanipulated, and three (60% [3 out of 5]) blastocysts were produced from
spindle-freeoocytes(0outof3).Theseobservationsareconsis- theseeggdonors(Figure4A).AfterplatingonmEFsandmanual
tent with recent conclusions that human MII oocytes undergo passaging,weestablishedtwostableNT-ESClines—onefrom
prematureactivationsecondarytospindleremoval(Tachibana eachoocytecohort(Figure4B).Thus,theseoutcomesconfirm
etal.,2013).Assumingthatmeiosis-specificfactorsareretained thereproducibilityofourhumanSCNTprotocols.
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wassimilarbetweenthesegroups,moreembryosderivedfrom
the R16 MII oocytes/cycle group arrested after the eight-cell
stagecomparedtotheothertwogroups(Figure5A).Inaddition,
thequalityofrecoveredSCNTblastocystscorrelatedinversely
with the number of collected oocytes per cycle. Whereas five
NT-ESC lines were derived from donors producing %10
oocytes/cycle, only one was recovered fromthe 11–15group,
andnocelllinewasestablishedfromcycleswithR16oocytes
(Figure5B).Thepeaksystemicestradiol(E2)levelineggdonors
priortohCGprimingpositivelycorrelatedwiththesubsequent
yield of oocytes (Figure S2). Thus, these observations imply
thathighnumbersofoocytescollectedfromovarianstimulation
protocolsareassociatedwithpooroocytequalityandreduced
reprogrammingabilityinthecontextofSCNT.
In an effort to define optimal stimulation protocols that are
compatiblewithhigh-qualityoocytesforSCNT,weanalyzedthe
impactofGnRHagonistandantagonistusedtosuppresspituitary
functionineggdonors.Priortoovarianstimulation,antimullerian
hormone (AMH) levels and antral follicle counts (AFC) were
measured for each egg donor (Table S3). Donors with higher
AMHandAFCprofilesindicativeofhighovarianreservereceived
GnRH agonist (Lupron, four cycles), and the remaining donors
receivedGnRHantagonist(Ganirelix,sixcycles)(TableS3).The
averagenumberofMIIoocytes(mean±SD)collectedpercycle
wasnotstatisticallydifferentbetweenthetwogroups(11.7±5.6
and20.5±11.9,respectively).However,SCNTembryodevelop-
mentbeyondtheeight-cellstagewassuboptimalforoocytespro-
ducedfollowingGnRHagonisttreatment(Figure5C).Moreover,all
sixNT-ESClineswerederivedexclusivelyfromoocytescollected
fromGnRHantagonist-treatedcycles(Figure5D).Basedonthese
observations,itispossiblethatpituitarysuppressionwithGnRH
Figure 4. Validation of Human SCNT with Nuclear Donor Cells
antagonist during ovarian stimulation may positively impact
DerivedfromaLeigh’sDiseasePatient
oocytereprogrammingcapability,makingthemcompatiblewith
(A)InvitrodevelopmentofSCNTembryosproducedwithskinfibroblastcells
fromaLeigh’sdiseasepatientandtwodifferenteggdonors(eggdonorsB SCNTblastocystdevelopmentandESCisolation.
andC).FifteenMIIoocyteswereretrievedfromeggdonorB,whereasonlyfive Lastly,welookedatwhetherpronuclearformationcanbeused
oocyteswerecollectedfromdonorC.SCNTblastocystsweregeneratedfrom asapredictivemarkerforSCNToutcomes.MostSCNTembryos
bothoocytecohorts. formedasinglepronucleusthedayafternucleartransfer(56%
(B)NT-ESCderivationefficiencyallowedisolationofonecelllinepereggdonor
[68outof122]),whereasasmallportion(20%[24outof122])dis-
cycle.
playedtwopronuclei(FigureS3).Asindicatedabove,pronuclear
SeealsoFiguresS2andS3.
formation was not observed in some one-cell SCNT embryos
(20% [24 out of 122]), whereas a minority (5% [6 out of 122])
RetrospectiveAnalysisofFactorsAffectingtheSuccess werealreadyatthetwo-cellstagebythetimeofexamination(Fig-
ofHumanSCNT ureS3).Afterseparateculture,wedeterminedthatcleavageand
Whereas SCNT manipulations and treatments were strictly early preimplantation development were similar among these
controlled,thequalityandquantityofhumanoocytesretrieved groups,andalthoughtheblastocystratewashigherinSCNTem-
from different egg donors varied significantly. High oocyte bryoswithtwopronuclei(39%),stableNT-ESClineswerepro-
numbersretrievedfromanovarianstimulationcyclearegener- ducedfromallfourembryotypes(FigureS3).Thus,despitethe
ally associated with poor clinical IVF outcomes (Pellicer et al., small number of SCNT embryos analyzed, it is reasonable to
1989;Santosetal.,2010;vanderGaastetal.,2006). concludethatvisiblepronuclearformationasconductedinthese
Therefore, weconducted a retrospective analysis of ovarian studiesdoesnotdirectlycorrelatewithNT-ESCderivation.
stimulation procedures and the number of oocytes retrieved
percycleversusSCNTembryodevelopmentandNT-ESCderi- AnalysisofHumanNT-ESCs
vationoutcomes.Wedividedoocytedonationcyclesintothree To confirm SCNT origin and define the degree of reprogram-
groups based on the range of collected mature MII oocytes— ming,weexpandedandextensivelyanalyzedthefourNT-ESC
specifically,10orfeweroocytespercycle(fivedonors),between lines derived from HDF-f fetal fibroblasts (designated as hE-
11and15oocytes(twodonors),andmorethan16oocytesper SO-NT1,hESO-NT2,hESO-NT3,andhESO-NT4).
cycle (three donors). Although survival after spindle removal, Initially, we employed microsatellite typing for 23 markers
fusion, pronuclear formation, and cleavage of SCNT embryos mapping 22 human autosomal loci and one X-linked locus for
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Figure5. OvarianStimulationandHumanSCNTOutcomes
(A)HumanSCNTdevelopmentvariedwiththenumberofoocytescollectedfromeachovarianstimulationcycle.Cyclesproducingtenorfeweroocyteswere
associatedwithimproveddevelopmentofSCNTembryos.
(B)TheefficacyofNT-ESCderivationalsopositivelycorrelatedwithfewernumbersofoocytescollectedintheovarianstimulationcycle.
(CandD)SCNTembryodevelopmentfromcyclestreatedwithGnRHagonistsorantagonists.Blastocystdevelopmentwashigherforoocytesrecoveredfrom
donorsreceivingaGnRHantagonist.NT-ESCswerederivedonlyfromoocytesrecoveredfromdonorsreceivingGnRHantagonist.
SeealsoFiguresS2andS3andTableS3.
nucleargenomegenotyping(Tachibanaetal.,2013).Theresults euploidfemalekaryotype(46XX)withnonumericalorstructural
unequivocally matched all four NT-ESC lines to the nuclear abnormalities(Figures6CandS4).
donorfetalfibroblasts,withnodetectablecontributionofoocyte ToassesspluripotencyinNT-ESClines,weinitiallyexamined
alleles(Figure6AandTableS4).ThedefiningfeatureofSCNTis expressionofgenericstemcellmarkersbyimmunocytochem-
thatthemitochondrialgenome(mtDNA)inSCNTembryosandin istry(ICC)andcomparedtheresultstotwoIVF-derivedESClines
NT-ESCsislargelycontributedbytheoocyte.Asexpected,anal- (hESO-7and-8).ThesecontrolESClinesandthefourNT-ESC
ysisofmtDNAsequenceswithinthedisplacementloop(Dloop) lineswereestablishedfromoocytesdonatedbythesamedonor
containingthehypervariablesegment(HSV)confirmedthatNT- (eggdonorA),andthustheycarriedidenticalmtDNA(Tachibana
ESC lines inherited mainly oocyte mtDNA (Figure 6B). During et al., 2013). Similar to controls, all NT-ESC lines expressed
fusion of cytoplasts with nuclear donor fibroblasts, a small OCT-4,NANOG,SOX2,SSEA-4,TRA-1-60,andTRA-1-81(Fig-
amountofsomaticcellmtDNAiscotransferredintotheresultant ures6DandS5).Moreover,wheninjectedintoimmunodeficient
embryosthatmayresultinheteroplasmy.Weemployedsensi- SCIDmice,allNT-ESClinesproducedtumorscontainingtissue
tive ARMS-qPCR (amplification refractory mutation system- andcelltypesrepresentingallthreegermlayers(Figure6E).An
quantitativepolymerasechainreaction)anddetectedalowlevel invitrodifferentiationassaydemonstratedefficientformationof
of somatic cell mtDNA in all four NT-ESC lines (3.4% ± 1.7%; embryoid bodies in suspension culture that, after attachment,
range1.2%–4.9%)(TableS5).Interestingly,thiscarryoverwas formedspontaneouslycontractingcardiomyocytes(MovieS1).
higher than what we had previously observed in ST-ESC lines Lastly, we conducted microarray expression analysis of the
(0.6% ± 0.9%) derived after spindle transfer between oocytes hESO-NT1 cell line and compared results to the IVF control,
(Tachibana et al., 2013). Cytogenetic analysis by G-banding hESO-7,andparentalsomaticcellsHDF-fusingtheAffymetrix
analysisindicatedthatallfourNT-ESClinescontainedanormal PrimeView platform. Initially, three biological replicates within
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Figure6. Genetic,Cytogenetic,andPlurip-
otencyAnalysisofHumanNT-ESCs
(A)NuclearDNAgenotypingfromfourhumanNT-
ESC lines (hESO-NT1, hESO-NT2, hESO-NT3,
and hESO-NT4) determined by microsatellite
parentage analysis. A total of 24 microsatellite
markerswereusedforeachanalysis.Therepre-
sentativemarkersforD2S1333andD4S413loci
demonstrate that the nuclear DNA in these cell
lines was exclusively derived from the somatic
HDF-fcellline.Nocontributionofoocytenuclear
DNAwasdetected.
(B) mtDNA genotyping by Sanger sequencing
demonstratedthatallNT-ESClinescontainoocyte
mtDNA.
(C) Cytogenetic G-banding analysis confirmed
thatallNT-ESCsexhibitedanormal46XXkaryo-
type(hESO-NT1resultisrepresentative).
(D)HumanNT-ESCsexpressedstandardplurip-
otency markers detected by immunocytochem-
istryforantibodiesagainstOCT4,NANOG,SOX2,
SSEA-4, TRA-1–60, and TRA-1–81. Original
magnification,3200;Ph,phasecontrast.
(E)Histologicalanalysisofteratomatumorspro-
ducedafterinjectionofhumanNT-ESCsintoSCID
mice.Anarrowandarrowheadinthetoppanel
indicateintestinal-typeepitheliumwithgobletcells
(endoderm)andcartilage (mesodermal),respec-
tively.Anarrowandarrowheadinthelowerpanel
depictneuroecto-dermal(ectoderm)andmuscle
(mesoderm)tissues,respectively.Originalmagni-
fication,3200.
SeealsoFiguresS4andS5andTablesS4andS5.
in this list (Tables S7 and S8). Interest-
ingly,theHLA-Cmajorhistocompatibility
genewashighlydownregulatedinhESO-
NT1 compared with hESO-7 (79-fold)
(TableS8).
DISCUSSION
Wedemonstratehereforthefirsttimethe
successfulreprogrammingofhumanso-
eachsamplewerecomparedagainsteachother.Forcompari- matic cells into ESCs following SCNT. By systematic analysis
sons, the detected signal for each probe set was plotted in a ofSCNTprocedures,insomecasesinformedbystudiesinthe
scattergraph, and the correlation value was calculated. This rhesus monkey, we identified several steps, including spindle
assay demonstrated 99% transcriptional correlation within removal,donorcellfusion,andcytoplastactivation,thatarecrit-
each cell type, suggesting that minimal variations existed ical for cellular reprogramming and SCNT blastocyst develop-
between biological replicates collected from different culture ment. Previous studies have indicated that meiotic arrest in
plates (Figure S6). Each NT-ESC and IVF-ESC type was humanMIIoocytesisunstablesuchthatintrusivemanipulations
comparedagainsteachotherandagainstsomaticcells(HDF-f). can induce rapid exit from the metaphase stage (Tachibana
Asexpected,bothstemcelltypesdisplayedlowtranscriptional et al., 2013). However, successful integration into and reprog-
correlationtofibroblasts(Figures7Aand7B).Among50genes ramming of interphase (G0/G1) somatic cell nuclei in MII
withthe highest fold change, many knownpluripotency genes cytoplastsarecriticallyimportantanddependentonnuclearre-
wereobserved,includingLIN28,POU5F1,NANOG,andSOX2 modeling events associated with high meiotic kinase activities
(Table S6). In contrast, ESCs derived by IVF and SCNT were present in cytoplasts (Choi and Campbell, 2010; Egli et al.,
similartoeachother(Figures7Aand7B).Sometranscriptional 2008;Mitalipovetal.,2007).Meioticcytoplastsinducerapidnu-
differences between human NT-ESCs and IVF-ESCs were clearenvelopebreakdown(NEBD)andprematurechromosome
observed.Howevernoknownpluripotencygeneswereincluded condensation (PCC) in transplanted interphase nuclei and
Cell153,1–11,June6,2013ª2013ElsevierInc. 7
Pleasecitethisarticleinpressas:Tachibanaetal.,HumanEmbryonicStemCellsDerivedbySomaticCellNuclearTransfer,Cell(2013),
http://dx.doi.org/10.1016/j.cell.2013.05.006
parthenogenetic development in intact MII oocytes (Mitalipov
etal.,2001),anditisgenerallyacceptedthat,ifartificialactiva-
tion supportsparthenogenetic embryodevelopment toblasto-
cystsandESCs,suchtreatmentsshouldbesufficienttoinduce
similar outcomes with reconstructed SCNT cytoplasts. How-
ever,inthepresentstudies,wefoundthatsupplementalelectro-
poration treatment was critical for cytoplast activation and
subsequent reprogramming was compatible with improved
SCNTdevelopment andESCderivation.Thus,itisreasonable
tospeculatethattherequirementsforoocyteorcytoplastactiva-
tioninSCNTandparthenogeneticsystemsaredifferent.
WealsoshowthathumanSCNTreprogrammingisdependent
onhumanoocytequality.Particularly,largernumbersofoocytes
retrieved following ovarian stimulation protocols with agonist
werenegativelycorrelatedwithhumanSCNTblastocystdevel-
opment.Thus,itisspeculatedthatovarianstimulationprotocols
routinelyutilizedforIVFtreatmentsmightbealteredsomewhatif
thegoalisobtainingESCderivationbySCNT.Conversely,sub-
optimal quality oocytes derived by in vitro maturation or other
sourcesarelikelytobeunsuitableforSCNT(S.M.,unpublished
data).Clearly,furtherstudiesaddressinggonadotropindosage
Figure7. MicroarrayExpressionAnalysisofHumanNT-ESCs andpituitarysuppressionregimensshouldbeevaluatedinthe
(A) Scatterplot analysis comparing expression profiles of human NT-ESCs contextofrecoveringhumanoocytessuitableforSCNT.
(hESO-NT1)withIVF-derivedESCcontrols(hESO-7)andparentalskinfibro-
Itisalsoimportanttonotethattheoocytequalityisultimately
blasts (HDF-f). Both IVF-ESCs and NT-ESCs displayed low transcriptional
linked to the genetic constitution of individual egg donors.
correlationtofibroblasts(leftandmiddle)butweresimilartoeachother(right).
(B)TreediagramanalysislinkingNT-ESCstoIVF-ESCs. Indeed,someoocytecohortsdidnotsupportSCNTblastocyst
SeealsoFigureS6andTablesS6,S7,andS8. developmentwiththepresentprotocol,whereasefficientblasto-
cyst formation (23% [5 out of 21]) has been reported using
differentprotocolsinvolvingelectrofusionfollowedbyactivation
convert them to spindle-like structures. It has been proposed with calcium ionophore and DMAP or DMAP/cytochalasin D
thatNEBDandPCCarerequiredforprecisecell-cyclesynchro- (Frenchetal.,2008).Nevertheless,reflectingwhatwepresumed
nizationbetweentheincominginterphasedonornucleusandthe tobeexceptionaloocytequalityfromonedonor,fiveblastocysts
recipientmitoticcytoplast(Eglietal.,2008).Suchdrasticchro- wereproducedfromjusteightoocytes,whichsubsequentlyre-
matintransformationsareassociatedwithefficientreprogram- sultedinthederivationoffourNT-ESClines.Interestingly,prior
ming and improved development of SCNT embryos, whereas eggdonationfromthisdonorwasalsoassociatedwithexcep-
lackoforincompletenuclearremodelingleadstoearlydevelop- tional outcomes that supported derivation of four ESC lines
mental arrest (Mitalipov et al., 2007; Nakajima et al., 2008; followingSTprocedures(Tachibanaetal.,2013).Althoughthe
Wakayamaetal.,1998;Wuetal.,2007). underlyinggeneticfactorscontributingtooocytequalityremain
Another issue is that somatic cell-specific transcription and unknown,certainFMR-1alleles,definedbytheCGGnucleotide
epigeneticfactorsmaintainingcellularidentitybecomedissoci- repeats,correlatewithimprovedoocytequalityandIVFsuccess
ated from the chromatin during NEBD and PCC and are more (Gleicheretal.,2011).Clearly,furtherstudiesarewarrantedto
actively replaced by oocyte-specific programs (Egli et al., elucidate the genetic and clinical parameters associated with
2008).Here,wesuggestthatretentionofmeioticactivityinhu- optimaloocytequalityforhumanSCNT.
man MII cytoplasts aided by enucleation in the presence of Givenpastdifficultiesinachievingtheultimategoalofproduc-
caffeine and HVJ-E-based fusion enhances conversion of so- inghumanNT-ESCs,itwasgenerallyassumedthatderivationof
matic cell nuclei to spindle-like structures. Moreover, human ESCsviaSCNTwouldrequireaninordinatenumberofoocytes
SCNTembryosgeneratedwithourapproachdevelopedtoblas- and thus could not be scaled up for widespread therapeutic
tocystsandNT-ESCs. use (Daley and Solbakk, 2011). However, here, our revamped
Anotherimportantfindinginourstudyisthatstandardactiva- SCNT protocols allowed derivation of at least one ESC line
tion treatments involving exposure to ionomycin and 6-DMAP fromeachoocytedonationcycle.
are not sufficient for supporting development of human SCNT AbatteryofpluripotencytestsperformedonhumanNT-ESCs
embryos.Duringnormalfertilization,spermentrytriggersoocyte demonstrated their similarities to genuine IVF-derived ESCs.
activation that is critical for completion of meiosis and for the Transcriptional interrogation indicated that NT-ESCs departed
initiationofmitoticdivisions.Activationisalsocriticalfortheoo- fromtheirparentalsomaticcellgeneexpressionpatternwithup-
cyte’scytoplasmtoacquirethereprogrammingandmetabolic regulation of pluripotency associated genes. In addition, NT-
activity that is necessary to support subsequent development ESCs demonstrated the ability to differentiate into a variety
(Susko-Parrish et al., 1994). Presently, the efficacy of artificial ofothercelltypesinteratomatumors.In-vitro-directeddifferen-
activation protocols is commonly measured by induction of tiation induced formation of contracting cardiomyocytes,
8 Cell153,1–11,June6,2013ª2013ElsevierInc.
Pleasecitethisarticleinpressas:Tachibanaetal.,HumanEmbryonicStemCellsDerivedbySomaticCellNuclearTransfer,Cell(2013),
http://dx.doi.org/10.1016/j.cell.2013.05.006
demonstratingtheirpotentialforregenerativemedicine.Genetic Products) or antagonist (Ganirelix, Merck) were given. Human chorionic
analysesshowedthatallfourNT-ESClinestestedtodatecon- gonadotropin(hCG)wasprescribedtotriggeroocytematuration.Self-admin-
tainednormaldiploidkaryotypes,withnodetectablegrosschro- istrationofinjectablerFSH(sc,Follistim,Merck)commencedoncycleday2
or 3 and continued for (cid:1)8–12 days. The starting gonadotropin dose was
mosomalabnormalitiesorcontributionfromtheoocytegenome
75–125IU/dayand1–2AhMG(sc,Menopur,FerringPharmaceuticals).The
apartfrommtDNA.
dosewasadjustedperindividualresponseusinganestablishedstepdown
Anapproachtopatient-specificpluripotentstemcellderiva- regimenuntilthedayofhCGinjection.Ovarianresponseandfolliculargrowth
tion that precludes the use of embryos is based on somatic weremonitoredbytransvaginalultrasoundandmeasurementofserumestra-
cellreprogrammingbyinducedexpressionofafewcriticaltran- diollevels.WhentwoormorefolliclesreachedR18mmindiameter,subjects
scription factors (referred as induced pluripotent stem cells receivedhCG(104IU,sc,Ovidrel,EMDSerono)totriggerfollicleandoocyte
maturation. Thirty-six hours following hCG injection, subjects underwent
[iPSCs]) (Takahashi et al., 2007; Takahashi and Yamanaka,
oocyteretrievalviatransvaginalfollicularaspiration.
2006; Yu et al., 2007). Recent studies have concluded that
Cumulus-oocyte complexes (COCs) were collected from aspirates and
humaniPSCsarecharacterizedbyhighfrequenciesofsubchro- placedinHTFw/HEPESmedium(LifeGlobal,IVFonline)supplementedwith
mosomal copy number alterations compared to IVF-derived 10%serumsubstitutesupplement(SSS;QuinnsAdvantageSerum,Cooper-
ESCs (Laurent et al., 2011). Some of these genetic changes Surgical)(HTFw/HEPES10%)at37(cid:3)C.COCsweretreatedwithhyaluronidase
wereassociatedwiththereprogrammingprocessitself,whereas to disaggregate cumulus and granulosa cells. Oocytes were isolated and
classifiedasgerminalvesicle(GV),meioticmetaphaseI(MI),andmaturemeta-
otherscouldhavebeeninheritedfromtheparentalsomaticcells
phaseII(MII)stageandwerethenplacedinGlobalmedium(LifeGlobal,IVFon-
(Laurentetal.,2011).Inaddition,iPSC-specificmethylationand line) supplemented with 10% SSS (Global 10%) at 37(cid:3)C in 5% CO2 and
transcriptionalabnormalitiesinimprintedregionsandXchromo-
coveredwithtissuecultureoil(SageIVF,CooperSurgical).
someswerealsodescribed(Nazoretal.,2012).Directcompar-
isonsbetweeniPSCsandNT-ESCsinthemouseindicatedthat NuclearDonorCellPreparations
such abnormalities are less frequent in the latter case, Commerciallyavailablefemaledermalfibroblastsoffetalorigin(HDF-f)were
concluding that SCNT-based reprogramming is more efficient obtainedfromScienCellResearchLaboratories,andLeighsyndromepatient
cellswereacquiredfromtheCoriellCellRepositories.Cellswereexpanded
in resetting the epigenetic identity of parental somatic cells
in75cm3cellcultureflasks(Corning)containingDMEM/F12supplemented
(Kim et al., 2010). Further comparisons of the genetic, epige- with100IUml(cid:4)1penicillin,100mgml(cid:4)1streptomycin(Invitrogen),10%FBS
netic, and transcriptional characteristics of human NT-ESCs, at37(cid:3)Cin5%CO.Fibroblastswerethendisaggregatedwithtrypsintreat-
2
IVF-ESCs,andiPSCsareclearlyjustified. mentandwerefrozendowninaliquotsof33105cellsinmediumcontaining
Lastly, one of the fundamental differences of SCNT-based 10%dimethylsulphoxide(DMSO,Sigma).Cellsweresubsequentlythawed
reprogramming is thatNT-ESCs containmtDNA almost exclu- priortoSCNTandculturedinfour-welldishes(Nunc)understandardcondi-
tionsuntiltheyreachedconfluency.Confluentcellsweresynchronizedinthe
sivelyoriginatingfromtheoocyte.Thisfactisgenerallyunderap-
G0/G1phaseofthecellcyclebycultureinDMEM/F12mediumwith0.5%
preciatedbutmayrepresentanadvantageoveriPSCderivation
FBSfor2–4daysbeforeSCNT.
becauseitensuresthatNT-ESCsacquirethepotentialtopro-
ducemetabolicallyfunctionalcellsandtissuesforcelltherapies, HumanSCNTProcedureandEmbryoCulture
irrespectiveofthenucleardonorcellmtDNA.Thus,SCNToffers Enucleationswereperformedasdescribedpreviously(Tachibanaetal.,2013).
astrategyforcorrectingofmtDNAmutationsandrescuingthe Oocyteswereplacedintoa50mlmanipulationdropletofHTFw/HEPES10%
metabolic function of pluripotent cells from patients with in- mediumcontaining5mg/mlcytochalasinBand1.25mMcaffeineinaglass-
bottomdish.Thedropletwascoveredwithtissuecultureoil,andoocytes
heritedoracquiredmtDNAdiseases.
were maintained at 37(cid:3)C for 10–15 min before spindle removal. The dish
wasthenmountedonthestageofaninvertedmicroscope(OlympusIX71)
EXPERIMENTALPROCEDURES equippedwithastagewarmer(http://www.tokaihit.com),Narishigemicroma-
nipulators, Oosight Imaging System (http://www.cri-inc.com), and a laser
RhesusMacaqueSCNT objective(http://www.hamiltonthorne.com).Anoocytewaspositionedusing
AllanimalprocedureswereapprovedbytheInstitutionalAnimalCareandUse aholdingpipettesothatthespindlewassituatedclosetothe2to4o’clock
CommitteeattheOregonNationalPrimateResearchCenter.Oocytecollec- position.Thezonapellucidanexttothespindlewasdrilledwithlaserpulses,
tions, SCNT, embryo culture, and NT-ESC isolation procedures were per- andanenucleationpipettewasinsertedthroughtheopening.Asmallamount
formed as previouslydescribed (Byrne et al., 2007; Sparman et al., 2009, ofcytoplasmsurroundedbyplasmamembraneandcontactingthespindle
2010). wasaspiratedintothepipette.Next,adisaggregatedfibroblastwasaspirated
intoamicropipetteandwasbrieflytransferredtothedropcontainingHVJ-E
HumanOocyteDonations extract(IshiharaSangyoKaisha).Thecellwasthenplacedintotheperivitelline
ThestudyprotocolswereapprovedbyboththeOHSUEmbryonicStemCell spaceofthecytoplastonthesideoppositethefirstpolarbody.Thisconstruct
ResearchOversightCommitteeandtheInstitutionalReviewBoard.Anony- wasrinsedwithHTFw/HEPES10%,transferredtoglobal10%medium,and
mouseggdonorsofages23–31wererecruitedthroughtheOHSUWomen’s incubatedat37(cid:3)Cin5%CO for30minuntilfusionoccurredasconfirmed
2
Health Research Unit via print and web-based advertising. Responding visuallybythedisappearanceofthedonorcellfromtheperivitellinespace.
womenwerescreenedwithrespecttotheirreproductive,medical,andpsy- Constructswerethensubjectedtoartificialactivationconsistingofelectropo-
chosocialhealth.Healthynonobese(BMI<28kg/m2)womenwhopassed rationpulses(two50msDCpulsesof2.7kVcm(cid:4)1)(ElectroSquarePorator
theinitialmedicalandpsychologicalevaluationswereinvitedtoparticipate T-820,BTX)in0.25Md-sorbitolbuffercontaining0.1mMcalciumacetate,
inaresearcheggdonationprogram.Eggdonorswerefinanciallycompen- 0.5mMmagnesiumacetate,0.5mMHEPES,and1mgml(cid:4)1fatty-acid-free
satedforthetime,effort,discomfort,andinconvenienceassociatedwiththe BSA. Activated SCNT constructs were then incubated in Global medium
donationprocess. (withoutserum)containing2mMDMAPat37(cid:3)Cin5%CO2for4hr.After
OvarianstimulationprotocolsfollowedestablishedclinicalIVFguidelinesas DMAP,SCNTembryoswererinsedwithHTFw/HEPES10%andtransferred
describedpreviously(Tachibanaetal.,2013).Inbrief,acombinationofrecom- into four-well dishes containing Global medium supplemented with 10%
binanthuman-follicle-stimulatinghormone(rFSH)andhumanmenopausalgo- FBS,12mMb-mercaptoethanol(BME),and10nMTrichostatinA(TSA,Sigma)
nadotropins (hMG) and either GnRH agonist (Lupron, Tap Pharmaceutical andculturedat37(cid:3)Cin5%CO2,5%O2,and90%N2for12hr.Embryoswere
Cell153,1–11,June6,2013ª2013ElsevierInc. 9
Pleasecitethisarticleinpressas:Tachibanaetal.,HumanEmbryonicStemCellsDerivedbySomaticCellNuclearTransfer,Cell(2013),
http://dx.doi.org/10.1016/j.cell.2013.05.006
thenrinsed,checkedforpronuclearformation,andculturedinGlobalmedium cleardonorcells.WearealsoindebtedtoVanessaDomush,ElizabethSmo-
supplementedwith10%FBSand12mMb-mercaptoethanol(BME)at37(cid:3)Cin lens, Cathy Ramsey, Ying Li, Riffat Ahmed, Brittany Daughtry, and Erin
5%CO2,5%O2,and90%N2foramaximumof7days.Themediumwas Wolfffortheirtechnicalsupport.Thehumanoocyte/embryoresearchwassup-
changedonlyonce,atday3ofculture. portedbyOHSUinstitutionalfundsandbythegrantfromLeducqFoundation.
ThenonhumanprimatestudiesweresupportedbygrantsfromtheNational
Isolation,Culture,andCharacterizationofHumanNT-ESCs Institutes of Health HD063276, HD057121, HD059946, EY021214, and
Afterzonapellucidaremovalviabriefexposureto0.5%protease(Sigma), 8P51OD011092.
SCNTblastocystswereplatedontoconfluentfeederlayersofmitomycin-C-
inactivated mouse embryonic fibroblasts (mEFs) and were cultured for Received:April30,2013
5–7daysat37(cid:3)C,3%CO,5%O,and92%N inESCderivationmedium Revised:May3,2013
2 2 2
consistingofDMEM/F12(Invitrogen)supplementedwith0.1mMnonessential Accepted:May3,2013
aminoacids,1mMl-glutamine,0.1mMb-mercaptoethanol,5ng/mlbasic Published:May15,2013
fibroblastgrowthfactor,10mMROCKinhibitor(Sigma),10%FBS,and10%
knockoutserumreplacement(KSR;Invitrogen).Beforeuse,freshESCderiva- REFERENCES
tionmediumwasmixed(50%:50%,v/v)withderivationmediumconditionedin
a24hrculturewithgrowinghumanESCs.Outgrowthsoftheinnercellmass Byrne,J.A.,Pedersen,D.A.,Clepper,L.L.,Nelson,M.,Sanger,W.G.,Gokhale,
(ICMs)weremanuallydissociatedintosmallclumpswithamicroscalpeland S.,Wolf,D.P.,andMitalipov,S.M.(2007).Producingprimateembryonicstem
werereplatedonfreshmEFplates.AfterthefirstpassageofICMoutgrowths, cellsbysomaticcellnucleartransfer.Nature450,497–502.
FBSandROCKinhibitorwereomitted,andKSRwasincreasedto20%.Col- Campbell,K.H.,McWhir,J.,Ritchie,W.A.,andWilmut,I.(1996).Sheepcloned
onieswithESC-likemorphologieswereselectedforfurtherpropagation,char- bynucleartransferfromaculturedcellline.Nature380,64–66.
acterization,andcytogeneticanalyses. Choi,I.,andCampbell,K.H.(2010).Treatmentofovineoocyteswithcaffeine
Immunocytochemistry,invivoandinvitrodifferentiation,andmicroarrayan- increasestheaccessibilityofDNaseItothedonorchromatinandreduces
alyseswereperformedasdescribed(Byrneetal.,2007;Mitalipovetal.,2007; apoptosisinsomaticcellnucleartransferembryos.Reprod.Fertil.Dev.22,
Tachibanaetal.,2013).DetailedprotocolsarealsoavailableintheExtended 1000–1014.
ExperimentalProcedures.
Daley, G.Q., and Solbakk, J.H. (2011). Stem cells: Triple genomes go far.
Nature478,40–41.
NuclearDNAGenotypingandCytogeneticAnalyses
Danan,C.,Sternberg,D.,VanSteirteghem,A.,Cazeneuve,C.,Duquesnoy,P.,
Genotyping of NT-ESCs was performed by microsatellite typing using 23
Besmond,C.,Goossens,M.,Lissens,W.,andAmselem,S.(1999).Evaluation
markersrepresenting22humanautosomallociandoneX-linkedlocus,aspre-
ofparentalmitochondrialinheritanceinneonatesbornafterintracytoplasmic
viously described (Tachibana et al., 2013). Karyotyping was performed by sperminjection.Am.J.Hum.Genet.65,463–473.
GWTbandingon20metaphasecellsfromeachhumanNT-ESClineattheHu-
Ding, X., Wang, Y., Zhang, D., Wang, Y., Guo, Z., and Zhang, Y. (2008).
manGeneticsLaboratory,UniversityofNebraskaMedicalCenter,asprevi-
Increasedpre-implantationdevelopmentofclonedbovineembryostreated
ouslydescribed(Tachibanaetal.,2013).
with5-aza-20-deoxycytidineandtrichostatinA.Theriogenology70,622–630.
Egli,D.,Birkhoff,G.,andEggan,K.(2008).Mediatorsofreprogramming:tran-
MitochondrialDNAGenotyping
scriptionfactorsandtransitionsthroughmitosis.Nat.Rev.Mol.CellBiol.9,
Genotyping of mtDNA was performed as previously described (Tachibana
505–516.
et al., 2013). The human mitochondrial displacement loop region (D loop)
harboringthehypervariablesegment1(HSV-1)wasamplifiedusingpublished Egli,D.,Chen,A.E.,Saphier,G.,Ichida,J.,Fitzgerald,C.,Go,K.J.,Acevedo,
primers(Dananetal.,1999).PCRproductsweresequenced,andtheinforma- N.,Patel,J.,Baetscher,M.,Kearns,W.G.,etal.(2011).Reprogrammingwithin
tive single nucleotide polymorphic (SNP) sites were identified using hoursfollowingnucleartransferintomousebutnothumanzygotes.Nat.Com-
Sequencherv.4.7software(GeneCodes).QuantitativemtDNAanalysiswas mun.2,488.
performedbyARMS-qPCRasdescribed(Tachibanaetal.,2013).Thedetailed Fan,Y.,Jiang,Y.,Chen,X.,Ou,Z.,Yin,Y.,Huang,S.,Kou,Z.,Li,Q.,Long,X.,
protocolisalsoavailableinExtendedExperimentalProcedures. Liu,J.,etal.(2011).Derivationofclonedhumanblastocystsbyhistonedeace-
tylaseinhibitortreatmentaftersomaticcellnucleartransferwithb-thalassemia
SUPPLEMENTALINFORMATION fibroblasts.StemCellsDev.20,1951–1959.
French,A.J.,Adams,C.A.,Anderson,L.S.,Kitchen,J.R.,Hughes,M.R.,and
SupplementalInformationincludesExtendedExperimentalProcedures,six Wood,S.H.(2008).Developmentofhumanclonedblastocystsfollowingso-
figures,eighttables,andonemovieandcanbefoundwiththisarticleonline maticcellnucleartransferwithadultfibroblasts.StemCells26,485–493.
athttp://dx.doi.org/10.1016/j.cell.2013.05.006. Gleicher,N.,Weghofer,A.,Lee,I.H.,andBarad,D.H.(2011).Associationof
FMR1genotypeswithinvitrofertilization(IVF)outcomesbasedonethnicity/
ACKNOWLEDGMENTS race.PLoSONE6,e18781.
Gurdon,J.B.(1962).Thedevelopmentalcapacityofnucleitakenfromintesti-
The authors would like to acknowledge the OHSU Embryonic Stem Cell nal epithelium cells of feeding tadpoles. J. Embryol. Exp. Morphol. 10,
Research Oversight Committee and the Institutional Review Board for 622–640.
providingoversightandguidancewithhumanembryoandESCstudies.We
Kim,K.,Doi,A.,Wen,B.,Ng,K.,Zhao,R.,Cahan,P.,Kim,J.,Aryee,M.J.,Ji,
thankeggdonorvolunteersandthestaffattheWomen’sHealthResearch
H.,Ehrlich,L.I.,etal.(2010).Epigeneticmemoryininducedpluripotentstem
UnitattheCenterforWomen’sHealth,theDivisionofReproductiveEndocri- cells.Nature467,285–290.
nology&InfertilityoftheDepartmentofObstetrics&GynecologyofOregon
Kishigami,S.,Mizutani,E.,Ohta,H.,Hikichi,T.,Thuan,N.V.,Wakayama,S.,
Health&ScienceUniversityfortheirsupportandprocurementofhumangam-
Bui,H.T.,andWakayama,T.(2006).Significantimprovementofmousecloning
etes.TheDivisionofAnimalResources,SurgeryTeam,AssistedReproductive
techniquebytreatmentwithtrichostatinAaftersomaticnucleartransfer.Bio-
Technology&EmbryonicStemCellCore,EndocrineTechnologyCore,and
chem.Biophys.Res.Commun.340,183–189.
Imaging&MorphologyCoreattheOregonNationalPrimateResearchCenter
providedexpertiseandservicesforthenonhumanprimatestudies.Hamilton Lanza,R.P.,Cibelli,J.B.,andWest,M.D.(1999).Prospectsfortheuseofnu-
ThornedonatedtheXYClonelasersystemforthisstudy.Wearegratefulto cleartransferinhumantransplantation.Nat.Biotechnol.17,1171–1174.
Dr.WarrenSangerandDiannaZaleskiforkaryotypingservices,Dr.CeciliaPe- Laurent,L.C.,Ulitsky,I.,Slavin,I.,Tran,H.,Schork,A.,Morey,R.,Lynch,C.,
nedoformicrosatelliteanalysis,andDr.CaryHardingforassistancewithnu- Harness,J.V.,Lee,S.,Barrero,M.J.,etal.(2011).Dynamicchangesinthe
10 Cell153,1–11,June6,2013ª2013ElsevierInc.