Human embryonic stem cells derived by somatic cell nuclear transfer

Please cite this article in press as: Tachibana et al., Human Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer, Cell (2013), Human Embryonic Stem Cells Derivedby Somatic Cell Nuclear Transfer Masahito Tachibana,Paula Michelle Sparman,Nuria Marti Rebecca Tippner-Hedges,Hong Eunju Alimujiang Fulati,Hyo-Sang Lee,Hathaitip Keith Masterson,Janine Larson,Deborah Karen David David Diana Jeffrey Jensen,Phillip Patton,Sumita Gokhale,Richard L. Stouffer,Don Wolf,and Shoukhrat 1Division of Reproductive & Developmental Sciences, Oregon National Primate Research Center, Oregon Health & Science University, 505NW 185th Avenue, Beaverton, OR 97006, USA2Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, Oregon Health & Science University, 3181 SW SamJackson Park Road, Portland, OR 97239, USA3Department of Oral Biology, Faculty of Dentistry, Mahidol University, Bangkok 10400, Thailand4Women's Health Research Unit, Oregon Health & Science University, 3303 SW Bond Avenue, Portland, OR 79239, USA5Boston University School of Medicine, 72 East Concord Street, Boston, MA 02118, USA6Present address: Laboratory Animal Center, Osong Medical Innovation Foundation, Chungbuk 363-951, Republic of Korea*Correspondence: cloning, the nature of reprogramming oocyte factors and theirmechanism of action remain largely unknown.
Reprogramming somatic cells into pluripotent em- In humans, SCNT was envisioned as a means of generating bryonic stem cells (ESCs) by somatic cell nuclear personalized embryonic stem cells from patients' somatic cells, transfer (SCNT) has been envisioned as an approach which could be used to study disease mechanisms and ulti- for generating patient-matched nuclear transfer (NT)- mately for cell-based therapies (; ESCs for studies of disease mechanisms and for ). However, the derivation of human nuclear transfer-em-bryonic stem cells (NT-ESCs) has not been achieved despite developing specific therapies. Past attempts to pro- numerous attempts during the past decade. The roadblock duce human NT-ESCs have failed secondary to early responsible for failure is early embryonic arrest of human embryonic arrest of SCNT embryos. Here, we identi- SCNT embryos precluding derivation of stable NT-ESCs. Typi- fied premature exit from meiosis in human oocytes cally, SCNT embryos fail to progress beyond the eight-cell stage, and suboptimal activation as key factors that are presumably due to an inability to activate critical embryonic responsible for these outcomes. Optimized SCNT genes from the somatic donor cell nucleus (; approaches designed to circumvent these limita- ). In a few cases, when SCNT embryos did reach tions allowed derivation of human NT-ESCs. When the blastocyst stage, either stable ESCs were not recovered or applied to premium quality human oocytes, NT- derivation was not attempted ( ESC lines were derived from as few as two oocytes.
). Though the underlying cause of early developmental NT-ESCs displayed normal diploid karyotypes and arrest remains unclear, most of these studies involving humanoocytes applied SCNT protocols developed for nonprimate spe- inherited their nuclear genome exclusively from cies. Previously, we demonstrated that SCNT procedures, when parental somatic cells. Gene expression and differ- adapted to primates, succeeded in reprogramming rhesus ma- entiation profiles in human NT-ESCs were similar to caque adult skin fibroblasts into NT-ESCs (; embryo-derived ESCs, suggesting efficient reprog- ). Therefore, we reasoned that, similar to ramming of somatic cells to a pluripotent state.
other mammals, human MII oocytes must contain reprogram-ming activity.
Several recent observations are relevant. Removal of human oocytes' nuclear genetic material (chromosomes) negatively im- Cytoplasmic factors present in mature, metaphase II (MII)-ar- pacts the cytoplast's subsequent ability to induce reprogram- rested oocytes have a unique ability to reset the identity of trans- ming (However, when a somatic cell nucleus planted somatic cell nuclei to the embryonic state. Since the is transplanted into an intact oocyte containing its own chromo- initial discovery in amphibians (), somatic cell nu- somes, the resulting polyploid embryos are able to develop to clear transfer (SCNT) success in a range of different mammalian blastocysts and support ESC derivation. One possible explana- species has demonstrated that such reprogramming activity in tion for these observations is that critical reprogramming factors enucleated or spindle-free oocytes (cytoplasts) is universal in human MII oocytes are physically associated with the chromo- somes or spindle apparatus and are depleted or critically dimin- However, despite numerous applications of SCNT for animal ished upon enucleation. Alternatively, it is possible that one or Cell 153, 1–11, June 6, 2013 ª2013 Elsevier Inc. 1

Please cite this article in press as: Tachibana et al., Human Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer, Cell (2013), Figure 1. Development of Monkey SCNTEmbryos Reconstructed with OptimizedProtocolsAlthough HVJ-E fusion was efficient, SCNT con-structs required activation by electroporation forblastocyst formation. Elimination of ionomycinfrom the activation treatment further improvedblastocyst development. I, ionomycin; DMAP,6-DMAP; CM, compact morula.
See also and virus of Japan (HVJ-E) to fuse nucleardonor cells with enucleated MII oocyteswhile maintaining cytoplasts in meiosis). Because oflimited oocyte availability, we first testedboth the feasibility and efficacy of HVJ- more of the steps in SCNT—namely, oocyte enucleation, donor E-based cell fusion on the development of rhesus macaque cell nucleus introduction, or cytoplast activation—negatively SCNT embryos.
impact cytoplast quality, rendering it incapable of inducing suffi- The fusion rate of adult fibroblasts with cytoplasts was 100% after HVJ-E treatment; however, and unexpectedly, the SCNT In considering distinct biological features of human oocytes embryos generated by HVJ-E fusion failed to progress beyond that could be involved, we focused on our recent observation the compact morula (CM) stage following standard ionomycin/ 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 monkey SCNT embryos produced by electrofusion developed Earlier, we suggested that retention of meiosis-spe- into blastocysts ; cific activities in the cytoplast is critical for nuclear remodeling af- Therefore, we postulated that exposure of the cytoplast to an ter transplantation of an interphase-stage somatic cell nucleus electropulse (electroporation) could be beneficial for SCNT re- (). This remodeling is positively correlated programming, perhaps as a supplemental activation stimulus.
with onward development of SCNT embryos after activation.
To test this possibility, we exposed HVJ-E-fused SCNT embryos Therefore, we systematically evaluated modifications in oocyte to electroporation before the standard I/DMAP activation treat- enucleation and donor cell introduction that might work to retain ment. Ten percent of SCNT embryos were capable of reaching meiosis factors in human cytoplasts. We also determined that the blastocyst stage Interestingly, this SCNT blasto- routine cytoplast activation treatments were insufficient to sup- cyst formation rate was unaffected even when exposure to port subsequent human SCNT embryo development. We initially ionomycin was omitted and SCNT embryos were activated used rhesus macaque oocytes to evaluate factors affecting suc- with electroporation followed by DMAP treatment cessful SCNT reprogramming in a primate system. Subse- Together, these results indicate that, although an electroporation quently, we refined SCNT approaches with high-quality human stimulus is not required for cell fusion, it is supportive of proper oocytes donated by healthy volunteers and demonstrated that cytoplast activation following SCNT.
methodological alterations significantly improve blastocyst for- Histone deacetylase inhibitors, such as trichostatin A (TSA), mation from human SCNT embryos. Moreover, we derived have been associated with improved SCNT reprogramming in several human NT-ESC lines from these embryos and validated several mammalian species (; that their nuclear DNA is an exclusive match to parental somatic ; We previously demonstrated enhanced cells, whereas mitochondrial DNA originated almost exclusively development of monkey SCNT embryos treated with 37.5 nM from oocytes. We also conducted extensive pluripotency assays TSA (from 4% up to 18% blastocyst development rate on human NT-ESCs to verify reprogramming.
]). However, blastocyst quality and potential to giverise to stable ESCs remained unknown. Here, we plated 16 mon- key SCNT blastocysts produced during TSA treatment on mitot-ically inactivated mouse embryonic fibroblast (mEF) feeders, but SCNT Protocol Optimization in a Nonhuman Primate none resulted in NT-ESC line isolation (We reasoned that, although TSA treatment was promoting blastocyst forma- Our recent studies demonstrated human MII oocyte sensitivity to tion, high TSA concentrations may negatively affect blastocyst premature activation induced by removal and reintroduction of quality and epiblast lineage integrity. Therefore, we tested meiotic spindles (and to the use of elec- several lower TSA concentrations, as well as shorter exposure trofusion in the context of cytoplast activation ( times, on monkey SCNT blastocyst development and ESC isola- Consequently, our present investigation began with opti- tion. Reducing the TSA concentration to 10 nM or shortening the mizing the use of envelope from inactivated hemagglutinating TSA exposure time from 24 to 12 hr did not affect blastocyst 2 Cell 153, 1–11, June 6, 2013 ª2013 Elsevier Inc.

Please cite this article in press as: Tachibana et al., Human Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer, Cell (2013), Figure 2. SCNT Blastocyst Development IsAffected by Premature Cytoplast Activation(A) Morphology of nuclear donor fetal fibroblastsbefore SCNT. (B) Poor-quality human SCNTblastocyst without distinct ICM produced withsuboptimal protocols; note the presence ofexcluded cells.
(C) Spindle-like structures detected when donornuclei were introduced into intact MII oocytes, butnot when introduction was conducted afterenucleation. Arrowhead and arrow point at thematernal MII spindle and somatic cell spindle,respectively.
(D) Somatic nuclear cell spindles were formed incytoplasts when oocyte enucleation and fusionwere conducted in the presence of caffeine.
(E) Human SCNT blastocyst with prominent ICM(asterisk) produced after caffeine treatment.
(F) NT-ESC colony with typical morphology derivedfrom a caffeine-treated SCNT human blastocyst.
dard ovarian stimulation protocols andtransvaginal follicular aspirations. Humandermal fibroblasts of fetal origin (HDF-f)synchronized in G0/G1 cell-cycle phasewere used as nuclear donors Spindle removal and HVJ-E-assisteddonor cell fusion were carried out within60 min of oocyte retrieval.
Most oocytes (95.2% [60 out of 63]) survived MII spindle removal conductedunder polarized microscopy (Oosight); ), and nuclear donor fibroblastswere introduced with 100% efficacy us-ing HVJ-E based fusion. Somatic cellnuclei did not form spindle-like structuresthat were detectable by noninvasive ex-amination under polarized microscopy.
Immediately after confirmation of fusion,oocytes were activated with electropora-tion/DMAP (4 hr) and exposed to 10 nMTSA for 12 hr. Most embryos (81.7% [49out of 60]; A, without caffeinegroup) formed one or two pronuclei atthe time of removal from TSA, whereasa slightly higher portion of embryoscleaved (86.7% [52 out of 60]), suggest- rates (However, only SCNT blastocysts produced with ing that some SCNT embryos did not exhibit visible pronuclei 10 nM TSA supported derivation of stable monkey NT-ESC lines, at the time of examination A). Most cleaved embryos though the number of plated blastocysts was small ().
developed to the eight-cell stage (61.5% [32 out of 52]), but We concluded that these optimized protocols in a nonhuman pri- few progressed to compact morula (13.5% [7 out of 52]) and mate model were adequate to serve as a starting point for further blastocyst (11.5% [6 out of 52]) stages Activation testing with human oocytes.
of embryonic genes and transcription from the transplanted so-matic cell nucleus are required for development of SCNT em- Producing Human SCNT Blastocysts and NT-ESC Lines bryos beyond the eight-cell stage ; Initially, human MII oocytes from healthy volunteers were ). Therefore, these results are consistent with the exposed to the SCNT approach that produced the best results premise that our modified SCNT protocol supports reprogram- in the nonhuman primate. Oocytes were retrieved following stan- ming of human somatic cells to the embryonic state.
Cell 153, 1–11, June 6, 2013 ª2013 Elsevier Inc. 3

Please cite this article in press as: Tachibana et al., Human Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer, Cell (2013), during spindle removal but decline due to spontaneous activa-tion, we focused on noninvasive treatments to maintain meioticarrest during manipulation.
We reported previously that the exposure of monkey oocytes to caffeine, a protein phosphatase inhibitor, was effective inprotecting the cytoplast from premature activation and improveddevelopment of SCNT embryos (). There-fore, human oocytes were maintained in 1.25 mM caffeine duringspindle removal and somatic cell fusion. As expected, somaticcell nuclei introduced into cytoplasts under these conditionsefficiently formed spindle-like structures that were detectableunder birefringence microscopy (83.3% [10 out of 12]) (D). More importantly, the blastocyst development rate ofcaffeine-treated embryos was notably enhanced (23.5%)compared to the standard SCNT group with caffeinegroup), and blastocysts were characterized by visible and prom-inent ICMs, similar to those observed for IVF-produced embryos( Remarkably, when eight SCNT blastocysts produced with caffeine exposure were utilized for ESCs isolation, all attachedto mEFs and four formed ICM outgrowths (whichgave rise to ESC-like colonies upon manual splitting onto freshmEF plates Subsequent passaging resulted in thepropagation of stable ESC colonies with typical morphologyand growth characteristics. This surprisingly high ESC derivationrate was similar to that reported in our previous study with humanIVF-derived blastocysts (50%) and was even higher than in Figure 3. Development of Human SCNT Embryos and NT-ESC manipulated spindle transfer embryos (38%) B) Derivation after Caffeine Treatment (A) Improved blastocyst development of human SCNT embryos treated with Collectively, our findings indicate that a protocol developed in caffeine. A total of 63 (five cycles) and 43 (three cycles) oocytes were utilizedfor SCNT without or with caffeine, respectively. Sixty (95.2%) and 42 (97.7%) the monkey model supported blastocyst development for hu- oocytes survived after SCNT micromanipulations.
man SCNT embryos. However, poor SCNT blastocyst quality (B) NT-ESCs were derived only from blastocysts produced with caffeine.
precluded ESC isolation. Subsequent incorporation of caffeine See also Figures S1, S2 and S3.
during enucleation and fusion allowed improved blastocystdevelopment and ESC line derivation.
Of note, human SCNT blastocysts exhibited poorly organized Reproducibility of Human SCNT Results trophectoderm and small or undetectable inner cell masses Interestingly, all four human NT-ESC lines were derived from oo- (ICMs). In addition, large blastomere-like cells were often cytes retrieved from one egg donor (egg donor A). Eight mature excluded B). Nevertheless, we plated six SCNT blasto- MII oocytes were recovered after a single stimulation cycle. Us- cysts onto feeder layers to examine their ability to support ESC ing fetal dermal fibroblasts as nuclear donor cells and following 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 produce stable ESC-like cells ( (80%) (Figure S1 available online). In the context of generating Though the observed SCNT blastocyst development rate was patient-specific pluripotent stem cells, reproducible results encouraging, further optimization of the human protocol focused with various patient-derived somatic cells and with different on improvements in embryo quality. To assess whether spindle egg donors are a necessity.
removal in human oocytes could cause spontaneous exit from We therefore acquired a skin fibroblast culture from a patient meiosis, we introduced somatic nuclei into intact MII oocytes with Leigh syndrome. A total of 15 and 5 MII oocytes were and examined their birefringence properties under polarized mi- collected from two unrelated egg donor volunteers (B and C) croscopy. All introduced somatic cell nuclei efficiently formed and were used for SCNT with these fibroblasts. All oocytes sur- spindle-like structures that were visible within 30 min of fusion vived spindle removal and successfully fused with nuclear donor (17 out of 17) Again, spindle formation was not cells. Following activation and culture, four (27%, [4 out of 15]) observed when somatic cell nuclei were fused with manipulated, and three (60% [3 out of 5]) blastocysts were produced from spindle-free oocytes (0 out of 3). These observations are consis- these egg donors After plating on mEFs and manual tent with recent conclusions that human MII oocytes undergo passaging, we established two stable NT-ESC lines—one from premature activation secondary to spindle removal each oocyte cohort (B). Thus, these outcomes confirm ). Assuming that meiosis-specific factors are retained the reproducibility of our human SCNT protocols.
4 Cell 153, 1–11, June 6, 2013 ª2013 Elsevier Inc.

Please cite this article in press as: Tachibana et al., Human Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer, Cell (2013), was similar between these groups, more embryos derived fromthe R16 MII oocytes/cycle group arrested after the eight-cellstage compared to the other two groups (A). In addition,the quality of recovered SCNT blastocysts correlated inverselywith the number of collected oocytes per cycle. Whereas fiveNT-ESC lines were derived from donors producing %10oocytes/cycle, only one was recovered from the 11–15 group,and no cell line was established from cycles with R16 oocytes(B). The peak systemic estradiol (E2) level in egg donorsprior to hCG priming positively correlated with the subsequentyield of oocytes (Figure S2). Thus, these observations implythat high numbers of oocytes collected from ovarian stimulationprotocols are associated with poor oocyte quality and reducedreprogramming ability in the context of SCNT.
In an effort to define optimal stimulation protocols that are compatible with high-quality oocytes for SCNT, we analyzed theimpact of GnRH agonist and antagonist used to suppress pituitaryfunction in egg donors. Prior to ovarian stimulation, antimullerianhormone (AMH) levels and antral follicle counts (AFC) weremeasured for each egg donor (). Donors with higherAMH and AFC profiles indicative of high ovarian reserve receivedGnRH agonist (Lupron, four cycles), and the remaining donorsreceived GnRH antagonist (Ganirelix, six cycles) ). Theaverage number of MII oocytes (mean ± SD) collected per cyclewas not statistically different between the two groups (11.7 ± 5.6and 20.5 ± 11.9, respectively). However, SCNT embryo develop-ment beyond the eight-cell stage was suboptimal for oocytes pro-duced following GnRH agonist treatment (C). Moreover, allsix NT-ESC lines were derived exclusively from oocytes collectedfrom GnRH antagonist-treated cycles D). Based on theseobservations, it is possible that pituitary suppression with GnRH Figure 4. Validation of Human SCNT with Nuclear Donor Cells antagonist during ovarian stimulation may positively impact Derived from a Leigh's Disease Patient oocyte reprogramming capability, making them compatible with (A) In vitro development of SCNT embryos produced with skin fibroblast cellsfrom a Leigh's disease patient and two different egg donors (egg donors B SCNT blastocyst development and ESC isolation.
and C). Fifteen MII oocytes were retrieved from egg donor B, whereas only five Lastly, we looked at whether pronuclear formation can be used oocytes were collected from donor C. SCNT blastocysts were generated from as a predictive marker for SCNT outcomes. Most SCNT embryos both oocyte cohorts.
formed a single pronucleus the day after nuclear transfer (56% (B) NT-ESC derivation efficiency allowed isolation of one cell line per egg donor [68 out of 122]), whereas a small portion (20% [24 out of 122]) dis- played two pronuclei (Figure S3). As indicated above, pronuclear See also Figures S2 and S3.
formation was not observed in some one-cell SCNT embryos(20% [24 out of 122]), whereas a minority (5% [6 out of 122]) Retrospective Analysis of Factors Affecting the Success were already at the two-cell stage by the time of examination (Fig- ure S3). After separate culture, we determined that cleavage and Whereas SCNT manipulations and treatments were strictly early preimplantation development were similar among these controlled, the quality and quantity of human oocytes retrieved groups, and although the blastocyst rate was higher in SCNT em- from different egg donors varied significantly. High oocyte bryos with two pronuclei (39%), stable NT-ESC lines were pro- numbers retrieved from an ovarian stimulation cycle are gener- duced from all four embryo types (Figure S3). Thus, despite the ally associated with poor clinical IVF outcomes ( small number of SCNT embryos analyzed, it is reasonable to conclude that visible pronuclear formation as conducted in these Therefore, we conducted a retrospective analysis of ovarian studies does not directly correlate with NT-ESC derivation.
stimulation procedures and the number of oocytes retrievedper cycle versus SCNT embryo development and NT-ESC deri- Analysis of Human NT-ESCs vation outcomes. We divided oocyte donation cycles into three To confirm SCNT origin and define the degree of reprogram- groups based on the range of collected mature MII oocytes— ming, we expanded and extensively analyzed the four NT-ESC specifically, 10 or fewer oocytes per cycle (five donors), between lines derived from HDF-f fetal fibroblasts (designated as hE- 11 and 15 oocytes (two donors), and more than 16 oocytes per SO-NT1, hESO-NT2, hESO-NT3, and hESO-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 Cell 153, 1–11, June 6, 2013 ª2013 Elsevier Inc. 5

Please cite this article in press as: Tachibana et al., Human Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer, Cell (2013), Figure 5. Ovarian Stimulation and Human SCNT Outcomes(A) Human SCNT development varied with the number of oocytes collected from each ovarian stimulation cycle. Cycles producing ten or fewer oocytes wereassociated with improved development of SCNT embryos.
(B) The efficacy of NT-ESC derivation also positively correlated with fewer numbers of oocytes collected in the ovarian stimulation cycle.
(C and D) SCNT embryo development from cycles treated with GnRH agonists or antagonists. Blastocyst development was higher for oocytes recovered fromdonors receiving a GnRH antagonist. NT-ESCs were derived only from oocytes recovered from donors receiving GnRH antagonist.
See also Figures S2 and S3 and nuclear genome genotyping The results euploid female karyotype (46XX) with no numerical or structural unequivocally matched all four NT-ESC lines to the nuclear abnormalities and S4).
donor fetal fibroblasts, with no detectable contribution of oocyte To assess pluripotency in NT-ESC lines, we initially examined alleles A and The defining feature of SCNT is expression of generic stem cell markers by immunocytochem- that the mitochondrial genome (mtDNA) in SCNT embryos and in istry (ICC) and compared the results to two IVF-derived ESC lines NT-ESCs is largely contributed by the oocyte. As expected, anal- (hESO-7 and -8). These control ESC lines and the four NT-ESC ysis of mtDNA sequences within the displacement loop (D loop) lines were established from oocytes donated by the same donor containing the hypervariable segment (HSV) confirmed that NT- (egg donor A), and thus they carried identical mtDNA ( ESC lines inherited mainly oocyte mtDNA During 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, and TRA-1-81 ( amount of somatic cell mtDNA is cotransferred into the resultant D and S5). Moreover, when injected into immunodeficient embryos that may result in heteroplasmy. We employed sensi- SCID mice, all NT-ESC lines produced tumors containing tissue tive ARMS-qPCR (amplification refractory mutation system- and cell types representing all three germ layers E). An quantitative polymerase chain reaction) and detected a low level in vitro differentiation assay demonstrated efficient formation of of somatic cell mtDNA in all four NT-ESC lines (3.4% ± 1.7%; embryoid bodies in suspension culture that, after attachment, range 1.2%–4.9%) ). Interestingly, this carryover was formed spontaneously contracting cardiomyocytes ( 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, (). Cytogenetic analysis by G-banding hESO-7, and parental somatic cells HDF-f using the Affymetrix analysis indicated that all four NT-ESC lines contained a normal PrimeView platform. Initially, three biological replicates within 6 Cell 153, 1–11, June 6, 2013 ª2013 Elsevier Inc.

Please cite this article in press as: Tachibana et al., Human Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer, Cell (2013), Figure 6. Genetic, Cytogenetic, and Plurip-otency Analysis of Human NT-ESCs(A) Nuclear DNA genotyping from four human NT-ESC lines (hESO-NT1, hESO-NT2, hESO-NT3,and hESO-NT4) determined by microsatelliteparentage analysis. A total of 24 microsatellitemarkers were used for each analysis. The repre-sentative markers for D2S1333 and D4S413 locidemonstrate that the nuclear DNA in these celllines was exclusively derived from the somaticHDF-f cell line. No contribution of oocyte nuclearDNA was detected.
(B) mtDNA genotyping by Sanger sequencingdemonstrated that all NT-ESC lines contain oocytemtDNA.
(C) Cytogenetic G-banding analysis confirmedthat all NT-ESCs exhibited a normal 46XX karyo-type (hESO-NT1 result is representative).
(D) Human NT-ESCs expressed standard plurip-otency markers detected by immunocytochem-istry for antibodies against OCT4, NANOG, SOX2,SSEA-4, magnification, 3200; Ph, phase contrast.
(E) Histological analysis of teratoma tumors pro-duced after injection of human NT-ESCs into SCIDmice. An arrow and arrowhead in the top panelindicate intestinal-type epithelium with goblet cells(endoderm) and cartilage (mesodermal), respec-tively. An arrow and arrowhead in the lower paneldepict neuroecto-dermal (ectoderm) and muscle(mesoderm) tissues, respectively. Original magni-fication, 3200.
See also Figures S4 and S5 and and in this list and Interest-ingly, the HLA-C major histocompatibilitygene was highly downregulated in hESO-NT1 compared with hESO-7 (79-fold)( We demonstrate here for the first time thesuccessful reprogramming of human so- each sample were compared against each other. For compari- matic cells into ESCs following SCNT. By systematic analysis sons, the detected signal for each probe set was plotted in a of SCNT procedures, in some cases informed by studies in the scattergraph, and the correlation value was calculated. This rhesus monkey, we identified several steps, including spindle assay demonstrated 99% transcriptional correlation within removal, donor cell fusion, and cytoplast activation, that are crit- 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 human MII oocytes is unstable such that intrusive manipulations compared against each other and against somatic cells (HDF-f).
can induce rapid exit from the metaphase stage ( As expected, both stem cell types displayed low transcriptional ). However, successful integration into and reprog- correlation to fibroblasts and 7B). Among 50 genes ramming of interphase (G0/G1) somatic cell nuclei in MII with the highest fold change, many known pluripotency genes cytoplasts are critically important and dependent on nuclear re- were observed, including LIN28, POU5F1, NANOG, and SOX2 modeling events associated with high meiotic kinase activities (In contrast, ESCs derived by IVF and SCNT were present in cytoplasts ; similar to each other (A and 7B). Some transcriptional ; ). Meiotic cytoplasts induce rapid nu- differences between human NT-ESCs and IVF-ESCs were clear envelope breakdown (NEBD) and premature chromosome observed. However no known pluripotency genes were included condensation (PCC) in transplanted interphase nuclei and Cell 153, 1–11, June 6, 2013 ª2013 Elsevier Inc. 7

Please cite this article in press as: Tachibana et al., Human Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer, Cell (2013), parthenogenetic development in intact MII oocytes ), and it is generally accepted that, if artificial activa-tion supports parthenogenetic embryo development to blasto-cysts and ESCs, such treatments should be sufficient to inducesimilar outcomes with reconstructed SCNT cytoplasts. How-ever, in the present studies, we found that supplemental electro-poration treatment was critical for cytoplast activation andsubsequent reprogramming was compatible with improvedSCNT development and ESC derivation. Thus, it is reasonableto speculate that the requirements for oocyte or cytoplast activa-tion in SCNT and parthenogenetic systems are different.
We also show that human SCNT reprogramming is dependent on human oocyte quality. Particularly, larger numbers of oocytesretrieved following ovarian stimulation protocols with agonistwere negatively correlated with human SCNT blastocyst devel-opment. Thus, it is speculated that ovarian stimulation protocolsroutinely utilized for IVF treatments might be altered somewhat ifthe goal is obtaining ESC derivation by SCNT. Conversely, sub-optimal quality oocytes derived by in vitro maturation or othersources are likely to be unsuitable for SCNT (S.M., unpublisheddata). Clearly, further studies addressing gonadotropin dosage Figure 7. Microarray Expression Analysis of Human NT-ESCs and pituitary suppression regimens should be evaluated in the (A) Scatterplot analysis comparing expression profiles of human NT-ESCs context of recovering human oocytes suitable for SCNT.
(hESO-NT1) with IVF-derived ESC controls (hESO-7) and parental skin fibro- It is also important to note that the oocyte quality is ultimately blasts (HDF-f). Both IVF-ESCs and NT-ESCs displayed low transcriptional linked to the genetic constitution of individual egg donors.
correlation to fibroblasts (left and middle) but were similar to each other (right).
(B) Tree diagram analysis linking NT-ESCs to IVF-ESCs.
Indeed, some oocyte cohorts did not support SCNT blastocyst See also Figure S6 and , and .
development with the present protocol, whereas efficient blasto-cyst formation (23% [5 out of 21]) has been reported usingdifferent protocols involving electrofusion followed by activation convert them to spindle-like structures. It has been proposed with calcium ionophore and DMAP or DMAP/cytochalasin D that NEBD and PCC are required for precise cell-cycle synchro- (Nevertheless, reflecting what we presumed nization between the incoming interphase donor nucleus and the to be exceptional oocyte quality from one donor, five blastocysts recipient mitotic cytoplast (Such drastic chro- were produced from just eight oocytes, which subsequently re- matin transformations are associated with efficient reprogram- sulted in the derivation of four NT-ESC lines. Interestingly, prior ming and improved development of SCNT embryos, whereas egg donation from this donor was also associated with excep- lack of or incomplete nuclear remodeling leads to early develop- tional outcomes that supported derivation of four ESC lines mental arrest (; following ST procedures ). Although the underlying genetic factors contributing to oocyte quality remain Another issue is that somatic cell-specific transcription and unknown, certain FMR-1 alleles, defined by the CGG nucleotide epigenetic factors maintaining cellular identity become dissoci- repeats, correlate with improved oocyte quality and IVF success ated from the chromatin during NEBD and PCC and are more (). Clearly, further studies are warranted to actively replaced by oocyte-specific programs elucidate the genetic and clinical parameters associated with Here, we suggest that retention of meiotic activity in hu- optimal oocyte quality for human SCNT.
man MII cytoplasts aided by enucleation in the presence of Given past difficulties in achieving the ultimate goal of produc- caffeine and HVJ-E-based fusion enhances conversion of so- ing human NT-ESCs, it was generally assumed that derivation of matic cell nuclei to spindle-like structures. Moreover, human ESCs via SCNT would require an inordinate number of oocytes SCNT embryos generated with our approach developed to blas- and thus could not be scaled up for widespread therapeutic tocysts and NT-ESCs.
use (). However, here, our revamped Another important finding in our study is that standard activa- SCNT protocols allowed derivation of at least one ESC line tion treatments involving exposure to ionomycin and 6-DMAP from each oocyte donation cycle.
are not sufficient for supporting development of human SCNT A battery of pluripotency tests performed on human NT-ESCs embryos. During normal fertilization, sperm entry triggers oocyte 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 initiation of mitotic divisions. Activation is also critical for the oo- from their parental somatic cell gene expression pattern with up- cyte's cytoplasm to acquire the reprogramming and metabolic 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 (Presently, the efficacy of artificial of other cell types in teratoma tumors. In-vitro-directed differen- activation protocols is commonly measured by induction of tiation induced formation of contracting cardiomyocytes, 8 Cell 153, 1–11, June 6, 2013 ª2013 Elsevier Inc.
Please cite this article in press as: Tachibana et al., Human Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer, Cell (2013), demonstrating their potential for regenerative medicine. Genetic Products) or antagonist (Ganirelix, Merck) were given. Human chorionic analyses showed that all four NT-ESC lines tested to date con- gonadotropin (hCG) was prescribed to trigger oocyte maturation. Self-admin- tained normal diploid karyotypes, with no detectable gross chro- istration of injectable rFSH (sc, Follistim, Merck) commenced on cycle day 2or 3 and continued for 8–12 days. The starting gonadotropin dose was mosomal abnormalities or contribution from the oocyte genome 75–125 IU/day and 1–2 A hMG (sc, Menopur, Ferring Pharmaceuticals). The apart from mtDNA.
dose was adjusted per individual response using an established stepdown An approach to patient-specific pluripotent stem cell deriva- regimen until the day of hCG injection. Ovarian response and follicular growth tion that precludes the use of embryos is based on somatic were monitored by transvaginal ultrasound and measurement of serum estra- cell reprogramming by induced expression of a few critical tran- diol levels. When two or more follicles reached R 18 mm in diameter, subjects scription factors (referred as induced pluripotent stem cells received hCG (104 IU, sc, Ovidrel, EMDSerono) to trigger follicle and oocytematuration. Thirty-six hours following hCG injection, subjects underwent oocyte retrieval via transvaginal follicular aspiration.
Recent studies have concluded that Cumulus-oocyte complexes (COCs) were collected from aspirates and human iPSCs are characterized by high frequencies of subchro- placed in HTF w/HEPES medium (LifeGlobal, IVFonline) supplemented with mosomal copy number alterations compared to IVF-derived 10% serum substitute supplement (SSS; Quinns Advantage Serum, Cooper- ESCs Some of these genetic changes Surgical) (HTF w/HEPES 10%) at 37C. COCs were treated with hyaluronidase were associated with the reprogramming process itself, whereas to disaggregate cumulus and granulosa cells. Oocytes were isolated andclassified as germinal vesicle (GV), meiotic metaphase I (MI), and mature meta- others could have been inherited from the parental somatic cells phase II (MII) stage and were then placed in Global medium (LifeGlobal, IVFon- (). In addition, iPSC-specific methylation and line) supplemented with 10% SSS (Global 10%) at 37C in 5% CO2 and transcriptional abnormalities in imprinted regions and X chromo- covered with tissue culture oil (Sage IVF, Cooper Surgical).
somes were also described ). Direct compar-isons between iPSCs and NT-ESCs in the mouse indicated that Nuclear Donor Cell Preparations such abnormalities are less frequent in the latter case, Commercially available female dermal fibroblasts of fetal origin (HDF-f) were concluding that SCNT-based reprogramming is more efficient obtained from ScienCell Research Laboratories, and Leigh syndrome patientcells were acquired from the Coriell Cell Repositories. Cells were expanded in resetting the epigenetic identity of parental somatic cells in 75 cm3 cell culture flasks (Corning) containing DMEM/F12 supplemented (). Further comparisons of the genetic, epige- with 100 IU ml1 penicillin, 100 mg ml1 streptomycin (Invitrogen), 10% FBS netic, and transcriptional characteristics of human NT-ESCs, at 37C in 5% CO2. Fibroblasts were then disaggregated with trypsin treat- IVF-ESCs, and iPSCs are clearly justified.
ment and were frozen down in aliquots of 3 3 105 cells in medium containing Lastly, one of the fundamental differences of SCNT-based 10% dimethyl sulphoxide (DMSO, Sigma). Cells were subsequently thawed reprogramming is that NT-ESCs contain mtDNA almost exclu- prior to SCNT and cultured in four-well dishes (Nunc) under standard condi-tions until they reached confluency. Confluent cells were synchronized in the sively originating from the oocyte. This fact is generally underap- G0/G1 phase of the cell cycle by culture in DMEM/F12 medium with 0.5% preciated but may represent an advantage over iPSC derivation FBS for 2–4 days before SCNT.
because it ensures that NT-ESCs acquire the potential to pro-duce metabolically functional cells and tissues for cell therapies, Human SCNT Procedure and Embryo Culture irrespective of the nuclear donor cell mtDNA. Thus, SCNT offers Enucleations were performed as described previously ).
a strategy for correcting of mtDNA mutations and rescuing the Oocytes were placed into a 50 ml manipulation droplet of HTF w/HEPES 10% metabolic function of pluripotent cells from patients with in- medium containing 5 mg/ml cytochalasin B and 1.25 mM caffeine in a glass-bottom dish. The droplet was covered with tissue culture oil, and oocytes herited or acquired mtDNA diseases.
were maintained at 37C for 10–15 min before spindle removal. The dishwas then mounted on the stage of an inverted microscope (Olympus IX71) EXPERIMENTAL PROCEDURES equipped with a stage warmer ), Narishige microma-nipulators, Oosight Imaging System (), and a laser Rhesus Macaque SCNT objective (An oocyte was positioned using All animal procedures were approved by the Institutional Animal Care and Use a holding pipette so that the spindle was situated close to the 2 to 4 o'clock Committee at the Oregon National Primate Research Center. Oocyte collec- position. The zona pellucida next to the spindle was drilled with laser pulses, tions, SCNT, embryo culture, and NT-ESC isolation procedures were per- and an enucleation pipette was inserted through the opening. A small amount formed as previously described (, of cytoplasm surrounded by plasma membrane and contacting the spindle was aspirated into the pipette. Next, a disaggregated fibroblast was aspiratedinto a micropipette and was briefly transferred to the drop containing HVJ-E Human Oocyte Donations extract (Ishihara Sangyo Kaisha). The cell was then placed into the perivitelline The study protocols were approved by both the OHSU Embryonic Stem Cell space of the cytoplast on the side opposite the first polar body. This construct Research Oversight Committee and the Institutional Review Board. Anony- was rinsed with HTF w/HEPES 10%, transferred to global 10% medium, and mous egg donors of ages 23–31 were recruited through the OHSU Women's incubated at 37C in 5% CO2 for 30 min until fusion occurred as confirmed Health Research Unit via print and web-based advertising. Responding visually by the disappearance of the donor cell from the perivitelline space.
women were screened with respect to their reproductive, medical, and psy- Constructs were then subjected to artificial activation consisting of electropo- chosocial health. Healthy nonobese (BMI < 28 kg/m2) women who passed ration pulses (two 50 ms DC pulses of 2.7 kV cm1) (Electro Square Porator the initial medical and psychological evaluations were invited to participate T-820, BTX) in 0.25 M d-sorbitol buffer containing 0.1 mM calcium acetate, in a research egg donation program. Egg donors were financially compen- 0.5 mM magnesium acetate, 0.5 mM HEPES, and 1 mg ml1 fatty-acid-free sated for the time, effort, discomfort, and inconvenience associated with the BSA. Activated SCNT constructs were then incubated in Global medium donation process.
(without serum) containing 2 mM DMAP at 37C in 5% CO2 for 4 hr. After Ovarian stimulation protocols followed established clinical IVF guidelines as DMAP, SCNT embryos were rinsed with HTF w/HEPES 10% and transferred described previously (In brief, a combination of recom- into four-well dishes containing Global medium supplemented with 10% binant human-follicle-stimulating hormone (rFSH) and human menopausal go- FBS, 12 mM b-mercaptoethanol (BME), and 10 nM Trichostatin A (TSA, Sigma) nadotropins (hMG) and either GnRH agonist (Lupron, Tap Pharmaceutical and cultured at 37C in 5% CO2, 5% O2, and 90% N2 for 12 hr. Embryos were Cell 153, 1–11, June 6, 2013 ª2013 Elsevier Inc. 9 Please cite this article in press as: Tachibana et al., Human Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer, Cell (2013), then rinsed, checked for pronuclear formation, and cultured in Global medium clear donor cells. We are also indebted to Vanessa Domush, Elizabeth Smo- supplemented with 10% FBS and 12 mM b-mercaptoethanol (BME) at 37C in lens, Cathy Ramsey, Ying Li, Riffat Ahmed, Brittany Daughtry, and Erin 5% CO2, 5% O2, and 90% N2 for a maximum of 7 days. The medium was Wolff for their technical support. The human oocyte/embryo research was sup- changed only once, at day 3 of culture.
ported by OHSU institutional funds and by the grant from Leducq Foundation.
The nonhuman primate studies were supported by grants from the National Isolation, Culture, and Characterization of Human NT-ESCs Institutes of Health HD063276, HD057121, HD059946, EY021214, and After zona pellucida removal via brief exposure to 0.5% protease (Sigma), SCNT blastocysts were plated onto confluent feeder layers of mitomycin-C-inactivated mouse embryonic fibroblasts (mEFs) and were cultured for Received: April 30, 2013 5–7 days at 37C, 3% CO Revised: May 3, 2013 2, 5% O2, and 92% N2 in ESC derivation medium consisting of DMEM/F12 (Invitrogen) supplemented with 0.1 mM nonessential Accepted: May 3, 2013 amino acids, 1 mM l-glutamine, 0.1 mM b-mercaptoethanol, 5 ng/ml basic Published: May 15, 2013 fibroblast growth factor, 10 mM ROCK inhibitor (Sigma), 10% FBS, and 10%knockout serum replacement (KSR; Invitrogen). Before use, fresh ESC deriva- tion medium was mixed (50%:50%, v/v) with derivation medium conditioned ina 24 hr culture with growing human ESCs. Outgrowths of the inner cell mass Byrne, J.A., Pedersen, D.A., Clepper, L.L., Nelson, M., Sanger, W.G., Gokhale, (ICMs) were manually dissociated into small clumps with a microscalpel and S., Wolf, D.P., and Mitalipov, S.M. (2007). Producing primate embryonic stem were replated on fresh mEF plates. After the first passage of ICM outgrowths, cells by somatic cell nuclear transfer. Nature 450, 497–502.
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