Reprogramming within hours following nuclear transfer into mouse but not human zygotes

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1 Reeive 22 Jun 2011 Aepte 8 Sep 2011 Pulishe 4 Ot 2011 DOI: /nomms1503 Reprogrmming within hours following nuler trnsfer into mouse ut not humn zygotes Dieter Egli 1,2,3,4, Alie E. Chen 1,2, Genevieve Sphier 1, Justin Ihi 1, Clire Fitzgerl 1, Kthryn J. Go 5, Niole Aeveo 5, Jy Ptel 5, Mnfre Betsher 1, Willim G. Kerns 6, Roin Goln 4, Ruolph L. Leiel 4, Dougls A. Melton 1,2,7 & Kevin Eggn 1,2,7 Fertilize mouse zygotes n reprogrm somti ells to pluripotent stte. Humn zygotes might therefore e useful for prouing ptient-erive pluripotent stem ells. However, logistil, legl n soil onsiertions hve limite the vilility of humn eggs for reserh. Here we show tht signifint numer of norml fertilize eggs (zygotes) n e otine for reprogrmming stuies. Using these zygotes, we foun tht when the zygoti genome ws reple with tht of somti ell, evelopment progresse normlly throughout the levge stges, ut then rreste efore the morul stge. This rrest ws ssoite with filure to tivte trnsription in the trnsferre somti genome. In ontrst to humn zygotes, mouse zygotes reprogrmme the somti ell genome to pluripotent stte within hours fter trnsfer. Our results suggest tht there my e previously unppreite rrier to suessful humn nuler trnsfer, n tht future stuies oul fous on the requirements for genome tivtion. 1 Deprtment of Stem Cell n Regenertive Biology, Hrvr University, Cmrige, Msshusetts 02138, USA. 2 Hrvr Stem Cell Institute, Hrvr University, Cmrige, Msshusetts 02138, USA. 3 The New York Stem Cell Fountion Lortory, New York, New York 10032, USA. 4 Nomi Berrie Dietes Center, College of Physiins n Surgeons, Columi University, New York, New York 10032, USA. 5 Reproutive Siene Center, Lexington, Msshusetts 02421, USA. 6 The Center for Preimplnttion Genetis, Rokville, Mryln 20850, USA. 7 Howr Hughes Meil Institute, Hrvr University, Cmrige, Msshusetts 02138, USA. Corresponene n requests for mterils shoul e resse to K.E. (emil: eggn@m.hrvr.eu).

2 nture ommunitions DOI: /nomms1503 The genertion of nimls y nuler trnsfer1,2 n the erivtion of humn emryoni stem ells 3 suggeste tht these two pprohes might e omine to generte ptientspeifi emryoni stem ell lines. Beuse they woul rry the ptient s genotype, suh stem ells might e useful for the proution of utologous trnsplnts 4,5 n for isese moelling 6. Stuies in mie hve emonstrte the potentil vlue of these omine pprohes for treting severe omine immune efiieny n Prkinson s isese 7,8. Although it is now possile to proue pluripotent stem ell lines with ptient genotypes using efine reprogrmming ftors 9,10, the equivleny of these ells to emryoni stem ells hs een questione. Severl groups hve foun ifferenes in gene expression, methyltion n ifferentition propensity etween ips ells n ES ells Further, it hs een suggeste tht inue pluripotent stem ells re not reprogrmme to the sme extent tht is oserve in emryoni stem ells following nuler trnsfer 16. Therefore, it remins importnt to pursue humn nuler trnsfer s n lterntive pproh for prouing stem ell lines. However, espite severl ttempts t humn nuler trnsfer 17 24, these efforts hve uniformly file to proue stem ell lines. Inste, most stuies reporte evelopmentl rrest uring the levge stges. Consistent with this, the only stem ell line purportely erive y humn nuler trnsfer 25 hs susequently een shown to originte from prthenogenesis rther thn reprogrmming of somti nuleus 26. We hve reently generte mouse emryoni stem ells y nuler trnsfer into mitoti zygotes 27, emonstrting tht these ell types might lso e useful supplement to ooytes for humn stuies. Here we report progrms for the quisition of humn preimplnttion emryos, resulting in the ontion of signifint numer of humn zygotes. When we performe nuler trnsfer, evelopment proeee through the levge stges ut rreste roun the time of omption. Our investigtion into the uses of this loke revele tht this evelopmentl rrest my e the result of filure to unergo trnsriptionl tivtion of the trnsferre somti hromosomes. Thus, there is rrier to reprogrmming following humn nuler trnsfer tht must e overome efore ptient-speifi emryoni stem ell lines n e erive. Results Dontion of humn zygotes. Beuse we hve h iffiulty in essing suffiient numers of unfertilize ooytes to proee with nuler trnsfer stuies 24, we onsiere the possiility tht humn zygotes oul serve s soure of reipient ytoplsm for nuler trnsfer. We therefore initite progrm for reruitment of ouples willing to onte their frozen zygotes for reserh. These protools were pprove y the prtiipting emi institutions ommittees on the use of humn sujets in reserh, stem ell reserh oversight ommittees n the Western Institutionl Review Bor, n Assoite of the Areittion of Humn Reserh Protetion Progrms. Between Jnury 2007 n My 2010, 4,061 in vitro fertiliztion (IVF) emryos were onte y ouples unergoing ssiste reproution. Though the mjority of these were ryopreserve t either the levge- or lstoyststges, 461 (11.3%) were frozen t the one-ell-stge n therefore useful for nuler trnsfer (Fig. 1). These humn zygotes were in exess of linil nee, ut otherwise norml. Nuler trnsfer into mitoti humn zygotes. For suessful reprogrmming, we h previously ulture interphse mouse zygotes until they entere the first mitosis, n only then rrie out nuler trnsfer 27. To investigte the reprogrmming potentil of humn zygotes, we thwe 386 frozen humn zygotes of whih Emryos onte 2623 Clevge stge 9 ND Zygotes Blstoysts In mitosis Lyse 17 Qulity of frozen zygotes 1PN PN 3PN Numer of zygotes Simultneous Hours More thn 1 y % Zygotes in mitosis BF γ -Tuulin β -Tuulin e 60 min 0 min 30 min 11 h f Hours Mirotuule irefringene Phospho H3 Merge Figure 1 Dontion of humn zygotes for stem ell reserh. () emryos onte for stem ell reserh. ND, not etermine; PN, pronuleus. () Mitoti entry time of humn zygotes (n = 107) in hours fter thw. Nuler envelope rekown of t lest one of the two pronulei ws efine s the time point of mitoti entry. Some zygotes were mitoti t the time of thw. () mitoti spinle formtion. Humn zygote 30 min fter nuler envelope rekown inluing rightfiel imge, mirotuule immunohistohemistry (rrowhes point to the entrosome t oth poles of the spinle) n spinle irefringene. Sle r left pnel 25 µm, right pnels 5 µm. ( f) synhrony in nuler envelope rekown. () Hours etween the rekown of the first pronuler envelope n the seon. (e) Zygote with synhronous pronuler envelope rekown. Numers inite the time from the rekown of the first nuler envelope. Sle r, 10 µm. The lotion of mitoti hromtin is irle. (f) 1PN zygote stine for phosphoryltion of Ser 27 on Histone H3, mrker of mitosis. Sle r, 10 µm.

3 nture ommunitions DOI: /nomms1503 ARTICLE Inhiition of spinle ssemly Enuletion Somti ell genome trnsfer Fusion pulse Inhiition of ytokinesis One-ell stge t two-ell timing Two-ell stge t four-ell timing Interphse zygote Zygote in prometphse Enuletion 5 min fter fusion pulse e GFP 2 h 30 min post fusion f GFP w/ noozole g Development GFP h i GFP j k l 7 h Mirotuule irefringene Figure 2 Somti ell nuler trnsfer into humn zygotes. () Shemti of nuler trnsfer into humn zygotes. () Zygote t 2PN stge in the presene of noozole. () Zygote t mitosis in the presene of noozole. Mternl n pternl hploi genomes re irle. () Pternl n mternl hploi genomes remove from the zygote. (e) Somti ell nuler trnsfer (green nuleus mrke y H2B:GFP) y fusion, (f) hromosome onenstion, (g) n 7 h fter exit from mitosis. (h) Spinle ssemly (i) hromosomes ligne t metphse. (j) Clevge, (k) six-ell stge, (l) morul-like, on 4 post fertiliztion. Sle r, 25 µm. 307 (80%) were vile. Of these, 259 (84.4%) ontine one mternl n one pternl pronuleus, s is typil for the first interphse in norml, iploi zygote (Fig. 1). These zygotes entere mitosis within 0 12 h fter thwing (Fig. 1), ssemle ipolr spinle (Fig. 1), n, of 28 ontrol zygotes, 9 (32%) evelope to the lstoyst stge (Supplementry Tle S1). To etter unerstn why not ll zygotes evelope to the lstoyst stge, n to exlue zygotes with low evelopmentl potentil from use in nuler trnsfer experiments, we nlyse the first mitosis in more etil. Another group hs previously reporte tht progression through the first mitosis n urtely preit evelopmentl potentil 28. Closer inspetion of zygotes entering into mitosis revele synhrony of nuler envelope rekown. 34/82 zygotes (41%) i not simultneously rek own their two pronulei, ut inste i so with ely of 30 min to up to 12 h (Fig. 1,e). When 1PN (pronuler) zygotes were stine for phosphoryltion of Histone H3, mitosis-speifi moifition of hromtin impose y AurorB kinse, we oserve extensive phosphoryltion on hromosomes of the pronuleus tht h roken own, ut not on hromtin of the pronuleus tht h retine nuler envelope (three of three zygotes) (Fig. 1f). In ition, we oserve vriety of other iiosynrsies in humn zygotes entering into mitosis. These inlue filure to onense the hromosomes (Supplementry Fig. S1), filure to ompletely issolve nuleoli (Supplementry Fig. S1) n filure to ssemle mitoti spinle (Supplementry Fig. S1). Zygotes exhiiting these normlities either rreste s single ells or eventully unerwent frgmenttion, n were exlue from use in nuler trnsfer. The unerlying use of normlities we oserve in frozen zygotes ws not entirely ler. There ws sustntil vrition in the ge of frozen zygotes onte to us, with the olest hving een ryopreserve for s mny s 20 yers (Supplementry Fig. S2). We o not fvour the ie tht these prolems re iret sie effet of the freeze-thw proess, s 3/3 humn zygotes frozen in mitosis were le to ssemle spinle within 30 min of thwing (Supplementry Fig. S3), n euse mouse zygotes frozen n thwe entere mitosis normlly n oul e use for nuler trnsfer (Supplementry Tle S2). In the ontext of erly nuler trnsfer experiments, whih ontrolle for miromnipultion of the humn zygote, we foun tht the mitoti spinle oul not properly iret ytokinesis if its position or orienttion ws isture (Supplementry Figs S4 S6). We therefore evise nuler trnsfer strtegy for humn zygotes tht ws nlogous to nuler trnsfer into ooytes, thus overoming this iffiulty (Fig. 2). In this novel nuler trnsfer strtegy, 69 humn interphse zygotes were ple into meium ontining the mirotuuleepolymerizing rug noozole (Fig. 2). In ll ses, these humn zygotes proeee into the first mitosis (Fig. 2), ut then, s intene, rreste ue to inhiition of spinle ssemly. This rrest in mitosis inue y noozole ws stle ut reversile n nontoxi (Supplementry Fig. S7), llowing us to perform miromnipultion. One mitoti entry ourre, we remove the zygoti hromosomes (Fig. 2) n introue interphse somti onor nulei (Fig. 2e). Skin ells of n ult helthy sujet, or n ult type 1 ieti sujet serve s nuler onors. Beuse nuler remoelling orreltes with reprogrmming in the rhesus monkey 29, we monitore the trnsferre somti hromtin hourly. We heke 46 of the 53 humn zygotes (53/69, 77%) tht survive these mnipultions n foun tht 43 h uner-

4 % Rehing evelopmentl stge Two-ell stge ZGA trnsripts 828 ZGA Clevge stge (3 8 ells) Blstoyst One-ell Morul y 1 y 4 e IVF mrna egrtion Amnitin 25 Nuler trnsfer Amnitin y 3& ZGA y 3&4 trnsripts up 46 ZGA trnsripts up 580 trnsripts normlly egre H2B-herry Nuler trnsfer 493 trnsripts normlly egre Figure 3 Developmentl potentil n ZGA fter nuler trnsfer into humn zygotes. () Developmentl potentil of ontrol IVF humn zygotes (lk olumns, n = 28), zygotes fter nuler trnsfer (lue olumns, n = 53), n zygotes ulture in the presene of lph-mnitin (re olumns, n = 23) isplye s the perentge of ells eveloping to n eyon the inite evelopmentl stge. () GFP expression fter humn nuler trnsfer. () Trnsgene retivtion fter mouse nuler trnsfer. Shown re ells 2 ys fter nuler trnsfer into mouse zygotes. () Genome tivtion fter humn SCNT. Venn igrms of genes upregulte > fivefol in the inite groups, (e) mternl mrna egrtion fter humn SCNT. Venn igrm of genes with trnsript levels reue y ftor of 5 or more in the inite groups. NT, nuler trnsfer. Sle r, 25 µm. gone nuler envelope rekown n hromosome onenstion within 6 h fter trnsfer (Fig. 2f; Supplementry Fig. S8). Therefore, we onlue tht erly nuler remoelling ws generlly suessful. As intene, the unreplite onor hromosomes oul not stisfy the spinle hekpoint n, therefore, these zygotes remine in mitosis (Supplementry Fig. S9). To inue mitoti exit, while inhiiting norml ytokinesis, we inute zygotes in the ylin-epenent protein kinse inhiitor purvlnol A, n the kinse inhiitor, 6-DMAP. Importntly, noozole, purvlnol A, n 6-DMAP re omptile with preimplnttion evelopment fter nuler trnsfer in mmmlin speies inluing the rhesus monkey 30 33, suggesting tht little, if ny, toxiity woul e expete when they re use. Within 4 h of rug tretment, 53/53 zygotes exite mitosis n entere the susequent interphse (Fig. 2g). Zygotes were then monitore for entry into the susequent mitosis, n within 30 h, 33/53 (62%) omplete the seon ell yle. On mitoti entry, 33/33 ssemle spinle (Fig. 2h,i), proeee through hromosome segregtion, susequent rouns of levge ivision (Fig. 2j,k), n 2/33 initite omption (Fig. 2l). However, we never oserve further evelopment to the lstoyst stge (n = 0/33). Genome tivtion fils fter trnsfer into humn zygotes. Beuse we oserve tht ll leving nuler trnsfer ells generte using norml humn zygotes (33/33) rreste t similr evelopmentl stge (Fig. 3), we wonere whether there ws funmentl loke to their evelopment. Beuse of two oservtions, we onsiere the hypothesis tht filure to tivte trnsription ws inhiiting evelopment fter nuler trnsfer. Our first oservtion ws tht green fluoresene from the H2B GFP protein trnsferre with the trnsgeni onor hromosomes ws etetle t the first interphse. However, fluoresene intensity erese with every levge n never returne (33/33 nuler trnsfer GFP 67 nture ommunitions DOI: /nomms1503 speimen oserve) (Fig. 3). This oservtion suggeste tht even the H2B GFP oing sequene uner ontrol of the strong CAAGS promoter ws never gin trnsrie. In ontrst, similr trnsgene ws routinely tivte fter mouse nuler trnsfer 31 (Fig. 3). Seon, we foun tht evelopment fter nuler trnsfer rreste t or shortly fter the stge, when mjor trnsriptionl tivtion in the emryo normlly ours 34,35. The steep inrese in trnsriptionl tivity in the norml emryo on y 3 fter fertiliztion, or fter the four-ell stge, ws lso terme zygoti genome tivtion (ZGA). We next ske whether there ws more glol efiit in trnsription following humn nuler trnsfer. To this en, we isolte RNAs from nuler trnsfer n ontrol emryos t equivlent evelopmentl stges (Supplementry Fig. S10) n then nlyse their trnsriptionl profiles using mirorrys. To unerstn the norml hnges in trnsription tht our uring erly stges of humn levge evelopment, we ompre the profiles of unmnipulte one-ell zygotes ollete on y 1 post IVF, to lstomeres of six to eight-ell emryos ollete on y 3 (~72 84 h post IVF) n y 4 of evelopment (~ h post IVF). Reltive to the mternl RNAs present in fertilize zygotes (n = 2 emryos), we foun tht 828 trnsripts were signifintly elevte ( > fivefol, P < 0.01, see Methos for sttistil nlysis) in ll IVF smples (n = 19 emryos, 4 iologil replites). This result suggests there is sustntil trnsriptionl tivity lrey ourring t the six- to eight-ell stge, s hs previously een reporte 35. In strk ontrst, when the profiles of evelopmentlly vne nuler trnsfer smples were exmine (n = 2, 2 inepenent iologil replites, 6- to 8-ell stge), only 101 (12%) of these 828 trnsripts were inrese in their unne reltive to the 1-ell zygote (Fig. 3) ( > fivefol, P < 0.01 for oth smples). As ontrol to ensure tht we oul urtely monitor trnsriptionl hnges in nuler trnsfer lstomeres, we lso monitore the ey of trnsripts. At the levge stge, we oserve tht 1,130 mternl trnsripts were sujet to ey in ll four ontrol smples ollete on y 3 n y 4 fter IVF. The levels of eh of these trnsripts eline y ftor of 5 or more uring norml evelopment from the 1-ell stge to the 6- to 8-ell stge ( < 0.2-fol, P < 0.01). Consistent with norml ey of sustntil frtion, ut not ll mternl RNAs, we foun tht 493 (44%) of these trnsripts were lso reue t the levge stge following nuler trnsfer ( < 0.2-fol, P < 0.01) (Fig. 3e). To exmine whether somti rther thn n emryoni gene expression progrm ws tive fter nuler trnsfer, we efine set of 2,153 trnsripts tht re trnsrie t higher levels in the somti onor ells thn in humn zygotes ( > tenfol, P < for 2 somti ell lines). Of the 2,153 trnsripts, 85 (4%) were elevte in nuler trnsfer smples ( > fivefol higher levels thn in zygotes, P < 0.01 for 2 smples), whers 350 (16%) were elevte in ontrol IVF emryos ( > fivefol higher levels thn in zygotes, P < 0.01 for 4 smples). Expression of these 350 trnsripts fter nuler trnsfer woul not require reprogrmming, s they re lrey tive in the somti onor ell. Nevertheless, only 50 of them (14%) were elevte fter nuler trnsfer. This inites tht neither n emryoni nor somti trnsriptionl progrm ws eing expresse, n even genes tht o not require reprogrmming file to e normlly expresse fter nuler trnsfer. Inhiition of trnsription inues levge stge rrest. Our trnsriptionl nlyses emonstrte tht there ws n unexpete filure to tivte trnsription t the pproprite time following humn nuler trnsfer. These results rise the possiility tht evelopment might rrest when the RNAs provie y the mternl stores re no longer suffiient to promote evelopment. If this hypothesis is orret, then ulture of norml fertilize zygotes in the trnsriptionl inhiitor lph-mnitin shoul e suffiient to

5 nture ommunitions DOI: /nomms1503 ARTICLE inue evelopmentl rrest ientil to tht oserve following nuler trnsfer. To test this ie, we ulture 23 zygotes for 5 ys in the presene of the trnsriptionl inhiitor lph-mnitin (Fig. 3). We oserve tht 22/23 of these zygotes leve n 10/23 evelope to the 6- to 8-ell stge (Supplementry Tle S1). However, no further evelopment to the lstoyst stge ourre. In these rreste lstomeres, the numer of trnsripts with elevte levels ws very similr to nuler trnsfer smples: only 46 of the 828 trnsripts (5.5%, P < 0.01) normlly rising in unne over the first 3 ys of evelopment were inrese > 5-fol over the sme time frme in lph-mnitin-trete smples (n = 2 speimen, 2 iologil replites) (Fig. 3). This result lso suggests tht s mny of the 101 ZGA trnsripts elevte in nuler trnsfer lstomeres my e ttriute to ifferentil messenger RNA stility rther thn tive trnsription. As oserve fter nuler trnsfer, tretment with lph-mnitin i not ompletely interfere with the egrtion of mternl RNAs. We foun tht 590 of 1,130 (47%, < 0.2- fol, P < 0.01) trnsripts tht normlly eline uring levge lso fell fter lph-mnitin tretment (smples ollete on ys 3 n 4 fter IVF) (Fig. 3e). Therefore, phrmologil inhiition of trnsription ws suffiient to inue hnges in mrna levels n evelopmentl rrest ientil to tht oserve fter humn nuler trnsfer. Reprogrmming within hours fter trnsfer into mouse zygotes. Although our experiments, thus fr, suggest tht there is loke to reprogrmming using humn zygotes tht is not present when mouse zygotes re use, our humn nuler trnsfer protool ws tehnilly istint from the pproh we h previously use for nuler trnsfer using mouse zygotes 27. To rule out the possiility tht tehnil spets of our humn nuler trnsfer proeure were preventing reprogrmming, we use methos tht we evelope here for humn nuler trnsfer n ske whether they oul support effiient evelopment n reprogrmming following nuler trnsfer in mouse zygotes. To monitor the stte of reprogrmming, we use til-tip firolsts rrying n Ot4 GFP trnsgene 36. In striking ontrst to our humn experiments, we oserve effiient evelopment to the morul n the lstoyst stges (signifine of ifferene mouse humn: χ 2 = 0.002, Chisqure-test) (Fig. 4; Supplementry Tle S3). We lso foun tht within less thn 36 h, GFP eme tivte from the somti hromosomes, suggesting tht reprogrmming of the Ot4 promoter h ourre n trnsription initite (Fig. 4). This reprogrmming following nuler trnsfer ws fr more rpi thn oserve following inution of pluripoteny in mouse firolsts using efine trnsription ftors 37,38 (Fig. 4). We lso ontrolle for the effets of ryopreservtion y performing nuler trnsfer into frozen-thwe mouse zygotes. These zygotes gve rise to lstoysts fter nuler trnsfer (Supplementry Tle S2). Thus, we n onlue tht even when preisely the sme nuler trnsfer methos re use, mouse zygotes supporte reprogrmming, wheres humn zygotes oul not. To more roly etermine whether trnsriptionl initition ws ourring normlly fter mouse nuler trnsfer into zygotes, we performe trnsriptionl profiling. In ontrst to the sitution in humn evelopment, where ZGA ours t the four- to eightell stge 35, in mouse, ZGA ours t the two-ell stge 39. Surprisingly, we foun tht trnsriptionl reprogrmming ws essentilly omplete y the en of the first ell yle, or h fter nuler trnsfer. Out of 1,025 trnsripts tht were upregulte etween ontrol mouse zygotes n the two-ell stge, 934 (91%) were lso upregulte fter nuler trnsfer ( > 5, P < 0.01) (Fig. 4). Chemilly mok-trete ontrol zygotes upregulte similr numer of trnsripts (898/1,025, 88%). Remrkly, of 179 trnsripts tht were upregulte t the two-ell stge reltive to the zygote ( > fivefol, P < 0.01) n tht were not expresse in til-tip firolsts, 151 were lso upregulte fter nuler trnsfer, n 154 in mok-trete ontrols. This level of reprogrmming ws ientil to tht oserve fter nuler trnsfer into mouse ooytes; the trnsriptome of nuler trnsfer emryos generte with zygotes One-ell t Two-ell timing Ot4::GFP tivtion Trnsfer 1, h 36 h Inhiition of ytokinesis 44 h 96 h No. of olonies 1,200 1, GFP Four ells Zygote NT t prometphse (1,266) IVF 98 Two-ell stge (1,025) 62 Mnipultion ontrol (1,019) Morul 0.5 Blstoyst 0.2 Correltion oeffiient Dys fter virl infetion Prthenote 2 ys post tivtion NT into ooyte 2 ys post tivtion NT into prometphse Zygote, 22 h post Two-ell stge* trnsfer Drug tretment ontrol* Prometphse zygote* Ooyte Figure 4 Trnsriptionl reprogrmming within hours fter mouse nuler trnsfer. () Ot4::GFP reprogrmming fter somti ell nuler trnsfer into mouse zygotes. () Numer of Ot4 GFP + olonies uring mouse ips genertion. Kinetis of Ot4::GFP retivtion in somti ells fter trnsution with retroviruses rrying the reprogrmming ftors Ot4, Sox2, Klf4 n -My. Dy 8 is the eighthth y fter the first exposure of somti ells to virl vetors. (,) ZGA n reprogrmming 22 h fter nuler trnsfer into mouse zygotes. () Venn igrm of trnsripts elevte t the two-ell stge. () luster igrm of the glol gene expression profile fter nuler trnsfer into ooytes or zygotes. *From ref. 31. Sle r, 10 µm.

6 nture ommunitions DOI: /nomms1503 Prometphse Interphse onor ells H2B-herry Anphse zygote Chromosome removl f NT into prometphse Zygote, 22h post trnsfer Two-ell stge Chromosome onenstion Conense hromtin 0 h HMC/ HMC/ 3 h post trnsfer Correltion oeffiient Genome-less ell* NT into zygote t nphse Zygote in mitosis Chromosome segregtion Continution of ell yle Reforme pronulei 2 h 4 h 12 h e HMC/ 20 h post trnsfer HMC/ g Two-ell stge (1,025) Genomeless (225) NT t nphse (550) 0.1 h % Morul n lstoysts /95 NT t prometphse NT t nphse Morul Blstoyst 0/69 Figure 5 Chromosome onenstion is require for evelopment n reprogrmming fter nuler trnsfer into mouse zygotes. () Shemti for nuler trnsfer into prometphse n () orresponing imges. Nulei of firolsts t interphse re trnsferre into zygote t prometphse of mitosis. Chromosomes re mrke with the re fluoresent fusion protein H2B-herry. Shown is the mitoti progression fter nuler trnsfer. Time inites the hours fter nuler trnsfer. Note the onenstion of hromosomes n their seprtion into two groups, followe y formtion of two pronulei fter inhiition of ytokinesis n entry into interphse. () Removl of the genome from zygote t nphse. () Nuler morphology fter trnsfer of interphse nulei. Smll inset: somti onor ell efore trnsfer. Note tht somti onor hromtin is not onense. (e) Nuler remoelling is slow n requires out 1 y to restruture the nuleus into lrge lstomere-like morphology. (f) Cluster nlysis of gene expression fter mouse SCNT. (g) Venn igrm of trnsripts elevte h fter nuler trnsfer t nphse. (h) Nuler trnsfer into zygotes t prometphse or t nphse of mitosis. Development to the morul n lstoyst stge s % of zygotes trnsferre with interphse somti ell nulei. Sle r, 10 µm. *From ref. 31. lustere losely with unmnipulte two-ell emryos, n nuler trnsfer emryos generte with ooytes lustere losely with prthenotes (Fig. 4). To etter unerstn the mehnism of reprogrmming in mouse zygotes, we trnsferre somti ells t vrious time points of mitosis. When somti nulei were trnsferre t prometphse, hromosome onenstion ourre within 2 h post trnsfer (Fig. 5,). In ontrst, when nulei were trnsferre t nphse of mitosis, hromosome onenstion i not our n nuler remoelling require 20 or more hours (Fig. 5 e). Reprogrmming n evelopment fter nuler trnsfer into mouse zygotes ws stritly epenent on nuler remoelling y hromosome onenstion. The trnsriptome of zygotes trnsferre t nphse lustere most losely with genome-less emryos, (Fig. 5f). Only 212/1,025 (20.7%) ZGA genes were normlly expresse fter nuler trnsfer t nphse (Fig. 5g), n of 179 ZGA genes silent in the somti onor ell, only 23 (12.8%) were normlly upregulte (Supplementry Fig. S11). Furthermore, ll emryos rreste t the two-ell stge when interphse nulei were trnsferre, ut evelopment to the lstoyst stge ws suessful when mitoti genomes were trnsferre. (Fig. 5h; Supplementry Tle S3). This oservtion rise the question whether filure to onense somti hromtin oul e responsile for the trnsriptionl n evelopmentl phenotype fter nuler trnsfer into humn zygotes. However, this ws not the se, s we foun tht 40/46 humn zygotes unerwent nuler envelope rekown n hromosome onenstion within 3 h fter trnsfer (Supplementry Fig. S8). Anorml kryotypes o not use trnsriptionl filure. It hs een suggeste tht mitoti normlities fter primte nuler trnsfer 40 use kryotypi errtions tht ontriute to evelopmentl rrest. We therefore use fluoresene in-situ hyriiztion to investigte whether similr normlities ourre fter humn nuler trnsfer n whether they might inue the trnsriptionl filures we oserve. Although some hromosome normlities were oserve, (Supplementry Fig. S12), normlities were lso foun in IVF lstomeres (Supplementry Tle S4), mny of whih ontinue evelopment to the morul n lstoyst stge. To iretly test whether or not kryotypi normlities oul e using trnsriptionl filures, we intentionlly inue neuploiy in otherwise norml fertilize ontrols, n then monitore their trnsriptionl tivity. To inue kryotypi normlities, we suppresse the first levge ivision, thus generting tetrploi ells with supernumerry entrosomes (Fig. 6). These ells forme multipolr spinles t the next mitosis n iretly leve into either three or four ells inste of two (Fig. 6; Supplementry Tle S5). As onsequene of the symmetri segregtion of hromosomes, the resulting lstomeres woul e expete to hve norml kryotypes. Despite their presumly norml kryotypes, we foun tht these lstomeres (n = 2, 1 replite) initite trnsription (489/828 ZGA genes (59%) were upregulte t lest fivefol, P < 0.001) (Supplementry Figure S13), n, therefore, their trnsriptionl profiles lustere losely together with norml IVF smples, n not with the nuler trnsfer smples (Fig. 6). Therefore, kryotypi normlities re not suffiient to explin the trnsriptionl filures we hve oserve following humn nuler trnsfer. Furthermore, s these zygotes were trete with noozole n the kinse inhiitors 6-DMAP n purvlnol A to inhiit ytokinesis, the sme rugs s in our nuler trnsfer protools, they ontrol for the effet of these rugs on trnsriptionl tivtion of the genome. We foun tht the trnsriptionl profile of these rug trete ontrol zygotes lustere with untrete ontrol emryos n lustere seprtely from nuler trnsfer emryos (Fig. 6). Therefore, the t suggests tht the rugs use re not iretly responsile for the trnsriptionl filures oserve fter somti ell nuler trnsfer.

7 nture ommunitions DOI: /nomms1503 ARTICLE Nuler trnsfer into zygotes with supernumerry pronulei. Humn zygotes fertilize y more thn one sperm 41, or whih o not omplete meiosis normlly, re routinely isre euse of their norml hromosome ontent. We hve shown tht mouse polyspermi zygotes n serve s ompetent reipient ells 27 n, therefore, ttempte to use 29 nlogous humn zygotes for nuler trnsfer n evelopmentl ontrols. As hs een oserve previously 42,43, when the evelopment of seven of these zygotes ws monitore, they unerwent norml ytokinesis (Supplementry Tle S5). We foun tht the unusul levge pttern tht resulte ws use y the nuletion of tripolr or tetrpolr mitoti spinles with supernumerry entrosomes t the spinle poles (Fig. 7). We hope tht y rresting these zygotes in mitosis (Fig. 7) n then removing their norml spinles, we might reple them with onor nulei tht woul support norml ytokinesis. After we trnsferre nulei into these mitoti zygotes, we gin oserve nuler envelope rekown n hromosome onenstion (Fig. 7). Following mitoti exit n inhiition of ytokinesis, entry into the seon interphse ourre normlly. However, in ll instnes, the next levge ivision ws multipolr (Supplementry Tle S5), resulting in more thn two, presumly neuploi Zygote in mitosis Bipolr spinle Centrosome Seon ell yle (interphse) Lte y 1 Suppression of levge One-ell t two-ell timing n entrosome replition Seon mitosis Dy 2 Dy 2 Multipolr spinle Telophse Multipolr levge Dy 2 Cleve RNA smples Zygote y 1 Zygote nuler trnsfer y 4 (6 ells) α-amnitin y 5 (6 8 ells) α-amnitin y 3 (6 8 ells) Drug-inue neuploiy (3 5 ells) IVF lte y 3 (6 8 ells) IVF erly y 4 (8 10 ells) Blstoyst Donor firolsts Donor firolsts Correltion oeffiient Mirotuule irefringene -Tuulin/ Figure 6 Filure to initite trnsription is not use y kryotypi normlities. () Inution of neuploiy in humn zygotes. Shemti showing evelopment fter suppression of levge t the first mitosis. () Development fter suppression of levge t the first mitosis. Arrows point to multipolr spinle s etete y mirotuule irefringene. Immunohistohemistry t nphse of mitosis shows the symmetri segregtion of hromtin to three poles (outer surfe of the leving ell is outline). Finl pnel: levge iretly to 4 ells. NT = nuler trnsfer. Dys inite the time post IVF. () Clustering of the trnsriptome of the inite smples. Sle r, 25 µm. β-tuulin Tetrpolr Tripolr % Zygotes in mitosis Kryoplst Fusion Chromosome onenstion 2 h Perientrin Hours post insemintion Mitosis Tripolr spinle Dy 2 Dy 2 Dy 2 Mitosis Tripolr levge Dy 2 Development Dy 3 Merge H2B-GFP β-tuulin/ Mirotuule irefringene H2B-GFP Figure 7 Nuler trnsfer into humn polyspermi zygotes. () Immunoytohemistry of ispermi humn zygotes t mitosis. Arrowhes point to perientrin positive entrosomes, rrows to tuulin-positive sperm tils. Sle r, 25 µm. () Entry of polyspermi zygotes into mitosis. Sle rs, left pnel 5 µm, mile n right pnels 25 µm. () Nuler trnsfer into humn polyspermi zygotes. Genome remove t mitosis, fusion with somti onor ell n hromosome onenstion of the somti ell genome 2 h post trnsfer re shown. () Clevge n evelopment fter nuler trnsfer into polyspermi zygotes. Time inites the ys post fertiliztion. The rrow points to irefringent spinle. Sle r, 25 µm.

8 nture ommunitions DOI: /nomms1503 ughter ells n evelopmentl rrest on y 3 fter fertiliztion (Fig. 7). Our results suggest tht we were unle to remove supernumerry entrosomes uring enuletion. Multipolr levge in humn tetrploi zygotes ws in striking ontrst to oth mouse n rit zygotes. In those speies, tetrploi or polyspermi zygotes me norml levge ivision, n effiiently evelope to the lstoyst stge (Supplementry Tle S6). These results suggest tht the entrosome hs more ominnt role in ireting spinle ssemly n ytokinesis in humn evelopment thn it oes in other speies. This oservtion hs sustntil rmifitions for humn nuler trnsfer n suggests tht, if more thn one entrosome is present in the zygote fter mnipultions re omplete, norml ptterns of spinle ssemly n levge will our. Disussion Here we report our ttempts to overome the logistil n sientifi ostles impeing the proution of ptient-speifi stem ell lines y nuler trnsfer. While it hs een iffiult to reruit ltruisti ooyte onors 24 we i suee in souring more thn 400 normlly fertilize eggs (zygotes) for our nuler trnsfer stuies. Development fter nuler trnsfer ws norml through the levge stges, ut, unlike IVF ontrols, ll leving nuler trnsfer ells rreste efore or t the time of omption with severe trnsriptionl normlities. An ientil phenotype oul e inue y inhiition of trnsription in IVF ontrols, ut not y the elierte inution of kryotypi normlities, suggesting tht trnsriptionl efets re more proximlly responsile for the evelopmentl rrest. Our finings re not the trivil result of smll smple size. Inste, our results of 160 nuler trnsfer experiments n more thn 175 ontrol mnipultions inite tht there is roust speies-speifi loke to reprogrmming tht must e overome efore humn stem ell lines n e erive. Our work n those of others suggests tht the evelopmentl rrest we oserve is not simply the result of using zygotes for nuler trnsfer s temporlly similr rrest is ommonly oserve fter nuler trnsfer into to humn unfertilize ooytes 18,20,25. Although single group hs reporte effiient trnsriptionl reprogrmming fter humn nuler trnsfer 20, they ompre somti ells to nuler trnsfer smples n their results re therefore onfoune y the presene of mternl mrnas, whih were not ppropritely ounte for y their nlyses. Another group hs generte single lstoyst fter trnsfer of emryoni stem ell nulei into humn ooytes 17, ut evelopment rreste when firolsts were trnsferre using ientil methos 18. This suggests tht evelopment n tivtion of the trnsferre genome epen on the epigeneti stte of the injete nuleus. In ontrst to humn zygotes, when we performe nuler trnsfer into mouse zygotes, we foun tht reprogrmming ws essentilly omplete within hours n inee within single ell yle. This result lso points to funmentl ifferene etween reprogrmming fter nuler trnsfer, n ips reprogrmming: t lest in nimls, nuler trnsfer meites n immeite trnsition from somti to pluripotent gene expression pttern, wheres reprogrmming y efine ftors seems to e grul proess, requiring ys or weeks 37,38. It is interesting to onsier tht this oul explin why stem ells generte y nuler trnsfer re inistinguishle 44 from stem ells erive from fertilize lstoysts, wheres in mouse 16,45 n humn 12,15,46 48 ips ell reprogrmming my t times e less omplete. Inomplete reprogrmming n evelopmentl efets fter somti ell nuler trnsfer hve een esrie in other verterte speies 36, For exmple, in ovine nuler trnsfer emryos, 3.8% of genes were foun to e inompletely reprogrmme 53. This is omprle to the results reporte here with mouse zygotes, where the mjority of trnsripts ( > 91%) re normlly expresse within 24 h fter nuler trnsfer. In ontrst, the trnsriptionl efets fter nuler trnsfer into humn zygotes exten fr eyon inomplete reprogrmming of few genes: the mjority of trnsripts, or more thn 88%, were not normlly expresse. Surprisingly, even genes tht were tive in the somti onor ell were not normlly expresse, suggesting filure to properly tivte the trnsferre somti ell genome. The speies-speifi ifferenes in trnsription fter nuler trnsfer might e ue to property of the humn egg, or of humn somti ells. Our results suggest tht investigting the requirements for trnsriptionl tivtion of the onor ell genome my help to overome the evelopmentl rrest ommonly oserve fter humn nuler trnsfer. Methos Nuler trnsfer into humn zygotes. Humn zygotes were otine using protools reviewe n pprove y the Committee on the Use of Humn Sujets (IRB) n the Stem Cell Reserh Oversight Committee (ESCRO) t Hrvr University. Humn zygotes were thwe with Quinn s vntge thw kit (Cooper Surgil). Zygotes were thwe in smll groups, from 6 12 for single experiment. On thwing, zygotes were wshe n ple in mirorops of Glol mei uner minerl oil, equilirte overnight t 37 C, 5% CO 2. The 307 zygotes surviving the thw were use s follows: 151 for nuler trnsfer experiments, 77 for other experimentl mnipultions, 12 for immunohistohemistry n RNA nlysis, 28 s evelopmentl ontrols, n the reminer for evelopmentl ontrols n the hrteriztion of zygoti mitosis. The following nuler trnsfer protool emerge s the most prtil: noozole ws e 1 2 h post thw t onentrtion of 50 µg ml 1. Lower noozole (Sigm) onentrtions (0.1 µg ml 1 ) were ineffetive for humn zygotes (Supplementry Fig. S14, Supplementry Tle S7), wheres vinlstine ws effetive even t low onentrtions (Supplementry Fig. S15). Without ition of noozole efore spinle removl, most spinle mteril ws remove with the zygoti genome (Supplementry Fig. S16), resulting in evelopmentl rrest n the formtion of norml spinles t the next mitosis (Supplementry Tle S1). On entry into mitosis, zygotes were stine with Hoehst (2 µg ml 1 ) (Sigm) for 5 10 min in GMOPS-plus ontining noozole. Zygotes were then ple on the hete stge in mirorops ontining 10 µg ml 1 CytoB n 50 µg ml 1 noozole to generte flui ytoplsm. A hole ws me in the zon pellui with XYClone Lser (Hmilton Thorne) using two to four µs pulses. The ws remove using fire-polishe pipette to prevent lysis (73/116 zygotes, 63%, survive the enuletion step). Chromosomes were visulize y low-intensity UV illumintion n/or Hoffmn moultion ontrst optis. Removl of genomi ws verifie y the presene of Hoehst/ omplex in the remove mteril n the sene in the zygote. A seon hole ws me n somti ell inserte elow the zon pellui. Somti ells were llowe to reh onflueny to inue ell yle rrest (for oth humn or mouse somti ells, this usully ourre within week fter pssge), n mintine s onfluent ultures until use for nuler trnsfer (etween 1 week to 2 months fter rehing onflueny). In some instnes, ells were expose to meium ontining 0.5% serum for 1 y efore use, for nuler trnsfer. Firolsts otine from mle ult T1D sujet (ID#1,011) or n ult helthy mle (ID#1,003) with norml kryotype (Supplementry Fig. S17) were use s nuler onors. Fusion of the somti ell ws one using two DC pulses of 1.6 kv m 1 of 20 µs with, using LF101 (Nep Gene) in ell fusion meium ontining 0.26 M mnnitol, 0.1 mm MgSO 4, 0.05% BSA, n 0.5 mm HEPES. 94 of 95 zygotes (99%) fuse uner these onitions, n none of them lyse. Using Piezo-meite injetion only 7/24, or 29% reovere from the lesion. On fusion, zygotes were returne to the inutor in Glol ulture meium. Chromosome onenstion ws monitore t lest every hour using GFP fluoresene. After 2 3 h, hromosome onenstion h ourre, n zygotes were stimulte to exit mitosis without levge using 2.5 mm 6-DMAP n 25 µm purvlnola (Sigm P4484), in Glol meium. After 4 5 h, when interphse nulei were pprent, zygotes were thoroughly wshe n then ulture in Glol meium supplemente with 15% plsmnte. Inution of zygotes in α-mnitin (Sigm) ws one eginning 3 4 h fter thw t onentrtion of 50 µg ml 1. Our totl survivl rte for the totl of 160 nuler trnsfer mnipultions ws 108/160 or 67%. Mirorry nlysis. For mirorry nlysis, totl RNA ws isolte using PioPure RNA isoltion kit (Moleulr Devies), n RNA ws mplifie y two to three rouns of T7 trnsription using the Illumin TotlPrep RNA Amplifition Kit. Amplifie RNA ws hyriize to the Illumin Sentrix humn gene expression BeChip RefSeq 8 v3.0 n re y the Illumin Be Arry Reer. Anlysis ws one using the Illumin Genome Stuio Progrm n Mirosoft Exel. Dt ws normlize to verge n ifferentil gene expression sore lulte in omprison to one-ell zygotes s referene. Fol hnge to one-ell zygotes were

9 nture ommunitions DOI: /nomms1503 ARTICLE lulte se on the verge signl for prtiulr trnsript using Mirosoft Exel. The verge fol hnge for two smples ws lulte (for exmple, two smples on 3 post IVF, two smples on 4 post IVF, two smples trete with mnitin, two NT smples n so on). If the verge fol hnge ws > 5 or < 0.2, n the ifferentil gene expression sore ws > 22 or < 22, orresponing to P-vlue of < 0.01 for oth smples, trnsript ws onsiere ifferentilly expresse. Differentil gene expression sore (Diffsore) is lulte y the Illumin Genome Stuio progrm using n Illumin ustom lgorithm. A iffsore of > 33 or < 33 orrespons to P-vlue of < All P-vlues inite in the mnusript were lulte in this mnner. The 828 ZGA trnsripts n the 1,130 mternl mrna ey trnsripts were t lest 5 ifferentilly expresse in the set of oth 3 n 4 IVF t points, eh onsisting of two smples. Nuler trnsfer into mouse zygotes n ooytes. Nuler trnsfer protools evelope on humn zygotes were use for nuler trnsfer into mouse zygotes. In rief, til-tip firolsts use for nuler trnsfer n ips genertion were otine from ult B6jBA-Tg (Pou5fI-EGFP)2Mnn/J mie. Mouse somti ells were prepre for nuler trnsfer s esrie ove for humn somti ells. Zygotes n ooytes were otine from BDF1 mie (Chrles River). Zygotes were rreste in mitosis y noozole n h their genome remove shortly (5 10 min) fter relese from noozole with or without the use of Hoehst/lowintensity UV illumintion. Nuler trnsfer ws one y two DC pulses of 1.6 kv m 1 in fusion uffer (71/98 or 72% of til tip ells fuse n integrte into the reipient zygote). Cytokinesis ws inhiite y inution inhiite y the sme omintion of kinse inhiitors s use for nuler trnsfer in humn zygotes: 10 µm of the ylin-epenent kinse inhiitor purvlnol A (Sigm P4484), 2 mm 6-DMAP (Sigm D2629), 0.5 µg ml 1 noozole, n optionlly with the ition of 20 µm of the uror B kinse inhiitor ZM (Toris Biosiene) for min. For nuler trnsfer t nphse, zygotes h their genome remove t nphse (40 50 min post-noozole relese) or, lterntively, the genome ws remove t prometphse, n zygotes were stimulte to enter nphse s ove (ytokinesis inhiitors purvlnol A, 6-DMAP n noozole) for min efore trnsfer. Control zygotes tht h ytokinesis inhiite with these sme ytokinesis kinse inhiitors effiiently evelope to the morul n lstoyst stge (nine lstoysts n one morul of ten zygotes), suggesting tht the omintion of these rugs is not toxi to mouse preimplnttion evelopment. Nuler trnsfer into mouse ooytes ws one s follows: ooytes were otine 14 h post hcg injetion, their spinle hromosome omplex ws remove in the presene of 5 µg ml 1 ytohlsinb. Til-tip firolsts were trnsferre into enulete ooytes y iret injetion, n tivte y 5 h inution in 1 mm SrCl2 in -free MZCB n 5 µg ml 1 ytohlsinb to inhiit polr oy extrusion, s esrie elsewhere 55,56. Arry t on nuler trnsfer into genome-less zygotes were otine from ref. 31. Drug-tretment-only ontrols were rreste s zygotes with noozole, n ytokinesis ws inhiite with the use of purvlnol A n 6-DMAP. For gene expression nlysis, RNA ws isolte from groups of ~20 ells, mplifie y 2 rouns of T7 polymerse trnsription n hyriize to Illumin rrys. Differentilly expresse genes were efine s hving Diffsore of > 22 or < thn 22 in two smples of the sme kin, n n t lest fivefol ifferene in the verge signl (verge of oth smples). Interphse zygotes were ollete h post hcg pulse, mitoti zygotes were otine y noozole-meite rrest until 33 h post hcg. ips genertion. Plt-E pkging ells were trnsfete (Fugene, Rohe) with retrovirl plsmis for Ot4, Sox2, Klf4 or -My n virl superntnt ws ollete, filtere, omine to ontin virus of ll four trnsription ftors, n ple on til-tip ells otine from ult B6jBA-Tg (Pou5fI-EGFP)2Mnn/J mie for 3 onseutive ys, s previously esrie (Ihi et l., 2009). The first pplition of virl superntnt ws ounte s y 0. Immunostining. Zygotes, lstomeres n lstoysts were fixe in 4% prformlehye overnight t 4 C, permeilize in PBS with 0.5%. Triton-X100 (PBS/T) for 20 min., loke in loking solution onsisting of 0.1% PBS/T with 10% FBS overnight t 4 C, inute in primry ntioy t 4 C in loking solution, then wshe for 1 h t room temperture in 0.1% PBS/T, inute with seonry onjugte ntioy in 0.1% PBS/T t room temperture for 1 h, wshe s ove, stine with Hoehst for 5 min n use for onfol imging. Ot4 ntioy (Snt Cruz, s5279) n Cx-2 ntioy (Biogenex) ws use t onentrtion of 1:200, n Brg-1 ntioy (Snt Cruz s-10768) ws use t onentrtion of 1:50. Conitions were mintine etween ifferent smples. BrU lelling ws one using the Amershm ell prolifertion kit (RPN20). Cells were inute into the lelling solution ilute 1:1,000 in KSOM for 18 h post trnsfer. Referenes 1. Guron, J. B. Ault frogs erive from the nulei of single somti ells. Dev. Biol. 4, (1962). 2. Wilmut, I., Shnieke, A. E., MWhir, J., Kin, A. J. & Cmpell, K. H. 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