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DOI: 10.1126/science.1094515, 1669 (2004);303 Science
, et al.Woo Suk Hwangfrom a Cloned BlastocystEvidence of a Pluripotent Human Embryonic Stem Cell Line Derived
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ological grouping of the genotypes through-out the epidemic (Fig. 2) (table S2).
In tracing the molecular evolution ofSARS-CoV in China, we observed that theepidemic started and ended with deletionevents, together with a progressive slowingof the nonsynonymous mutation rates and acommon genotype that predominated duringthe latter part of the epidemic. The mecha-nistic explanation for the selective adaptationand purification processes that led to suchgenomic evolutionary changes in SARS-CoVrequires further work (29). Nonetheless, thisstudy has provided valuable clues to aid fur-ther investigation of this remarkable evolu-tionary tale.
We have sequenced the complete S gene(GenBank accession number AY525636)from an oropharyngeal swab sample (sam-pling date, 22 December 2003) collectedfrom the most recent index patient of the cityof Guangzhou (onset date, 16 December2003; hospitalized 20 December 2003;www.wpro.who.int/sars/docs/pressreleases/pr_27122003.asp). Phylogenetic analysis ofthis S gene sequence with those from thehuman SARS-CoV and palm civet SARS-like coronavirus indicated that this most re-cent case of SARS-CoV is much closer to thepalm civet SARS-like coronavirus than toany human SARS-CoV detected in the pre-vious epidemic (fig. S7 and table S4). Be-cause it is evidently different from the recentlaboratory infections in Singapore (www.who.int/csr/don/2003_09_24/en) and Taiwan(www.who.int/mediacentre/releases/2003/np26/en), it strengthens the argument foranimal origin of the human SARS epidemic.
References and Notes1. R. A. Fouchier et al., Nature 423, 240 (2003).2. T. G. Ksiazek et al., N. Engl. J. Med. 348, 1953 (2003).3. C. Drosten et al., N. Engl. J. Med. 348, 1967 (2003).4. P. A. Rota et al., Science 300, 1394 (2003).5. M. A. Marra et al., Science 300, 1399 (2003).6. G. Vogel, Science 300, 1062 (2003).7. Y. J. Ruan et al., Lancet 361, 1779 (2003).8. Y. Guan et al., Science 302, 276 (2003).9. S. K. W. Tsui, S. S. C. Chim, Y. M. D. Lo, N. Engl. J. Med.
349, 187 (2003).10. S. S. C. Chim et al., Lancet 362, 1807 (2003).11. R. W. K. Chiu, S. S. C. Chim, Y. M. D. Lo, N. Engl.
J. Med. 349, 1875 (2003).12. J. S. Rest, D. P. Mindell, Infect. Genet. Evol. 3, 219 (2003).13. N. S. Zhong et al., Lancet 362, 1353 (2003).14. Supporting materials are available on Science Online.15. K. W. Tsang et al., N. Engl. J. Med. 348, 1977 (2003).16. N. Lee et al., N. Engl. J. Med. 348, 1986 (2003).17. Centers for Disease Control and Prevention, Morb.
Mortal. Wkly. Rep. 52, 241 (2003).18. E. J. Snijder et al., J. Mol. Biol. 331, 991 (2003).19. SARS-like coronaviruses were isolated from palm civ-
ets farmed domestically in Hubei Province, China, byHu et al. at the Wuhan Institute of Virology, ChineseAcademy of Sciences. Partial genome sequencing re-vealed an 82-nt deletion within the Orf8 region,which is identical to that found in human SARS-CoVisolates from the early patients of Zhongshan,Guangdong Province, China. Contamination can beruled out because no human SARS-CoV isolate withthe 82-nt deletion has ever been found in that insti-tute or has been isolated in that region of China.
20. The SARS-CoV sequence with the 415-nt deletion
(CUHK-LC2, CUHK-LC3, CUHK-LC4, and CUHK-LC5)was obtained from two SARS patients whose diseasewas linked to a late cluster of SARS cases in HongKong. Both patients had disease onset in mid-May2003. The CUHK-LC2 sequence was initially obtainedfrom the culture isolate of a throat wash specimen ofan infected hospital health care worker and was laterconfirmed from the same specimen directly. CUHK-LC3, CUHK-LC4, and CUHK-LC5 were obtained fromthree different nasal swab specimens both directlyand from the culture supernatants of an elderly pa-tient who acquired SARS in the same hospital.
21. M. M. C. Lai, K. V. Holmes, in Fields Virology, D. M.Knipe, P. M. Howley, Eds. (Lippincott Williams &Wilkins, New York, ed. 4, 2001), chap. 35.
22. E. G. Brown, H. Liu, L. C. Kit, S. Baird, M. Nesrallah,Proc. Natl. Acad. Sci. U.S.A. 98, 6883 (2001).
23. S. H. Seo, E. Hoffmann, R. G. Webster, Nature Med. 8,950 (2002).
24. D. Rasschaert, M. Duarte, H. Laude, J. Gen. Virol. 71,2599 (1990).
25. W.-H. Li, M. Tanimura, P. M. Sharp, Mol. Biol. Evol. 5,313 (1988).
26. J. W. Drake, J. J. Holland, Proc. Natl. Acad. Sci. U.S.A.96, 13910 (1999).
27. Z. Luo, A. M. Matthews, S. R. Weiss, J. Virol. 73, 8152(1999).
28. R. M. Bush, C. B. Smith, N. J. Cox, W. M. Fitch, Proc.Natl. Acad. Sci. U.S.A. 97, 6974 (2000).
29. P. W. Ewald, J. Urban Health 75, 480 (1998).30. See SOM Text at Science Online for
acknowledgments.
Supporting Online Materialwww.sciencemag.org/cgi/content/full/1092002/DC1Materials and MethodsSOM TextReferences and NotesFigs. S1 to S7Tables S1 to S4
29 September 2003; accepted 14 January 2004Published online 29 January 2004;10.1126/science.1092002Include this information when citing this paper.
Evidence of a Pluripotent HumanEmbryonic Stem Cell Line
Derived from a Cloned BlastocystWoo Suk Hwang,1,2* Young June Ryu,1 Jong Hyuk Park,3
Eul Soon Park,1 Eu Gene Lee,1 Ja Min Koo,4 Hyun Yong Jeon,1
Byeong Chun Lee,1 Sung Keun Kang,1 Sun Jong Kim,3 Curie Ahn,5
Jung Hye Hwang,6 Ky Young Park,7 Jose B. Cibelli,8
Shin Yong Moon5*
Somatic cell nuclear transfer (SCNT) technology has recently been used to generateanimals with a common genetic composition. In this study, we report the derivationof a pluripotent embryonic stem (ES) cell line (SCNT-hES-1) from a cloned humanblastocyst. The SCNT-hES-1 cells displayed typical ES cell morphology and cell surfacemarkers and were capable of differentiating into embryoid bodies in vitro and offorming teratomas in vivo containing cell derivatives from all three embryonic germlayers in severe combined immunodeficient mice. After continuous proliferation formore than 70 passages, SCNT-hES-1 cells maintained normal karyotypes and weregenetically identical to thesomaticnucleardonorcells.Althoughwecannotcompletelyexclude the possibility that the cells had a parthenogenetic origin, imprinting analysessupport a SCNT origin of the derived human ES cells.
The isolation of pluripotent human embry-onic stem (ES) cells (1) and breakthroughsin somatic cell nuclear transfer (SCNT) inmammals (2) have raised the possibility ofperforming human SCNT to generate po-tentially unlimited sources of undifferenti-
ated cells for use in research, with potentialapplications in tissue repair and transplan-tation medicine. This concept, known as“therapeutic cloning,” refers to the transferof the nucleus of a somatic cell into anenucleated donor oocyte (3). In theory, theoocyte’s cytoplasm would reprogram thetransferred nucleus by silencing all the so-matic cell genes and activating the embry-onic ones. ES cells would be isolated fromthe inner cell mass (ICM) of the clonedpreimplantation embryo. When applied in atherapeutic setting, these cells would carrythe nuclear genome of the patient; there-fore, it is proposed that after directed celldifferentiation, the cells could be trans-planted without immune rejection to treatdegenerative disorders such as diabetes,osteoarthritis, and Parkinson’s disease
1College of Veterinary Medicine, 2School of Agricul-tural Biotechnology, Seoul National University, Seoul151-742, Korea. 3Medical Research Center, MizMediHospital, Seoul, 135-280, Korea. 4Gachon MedicalSchool, Incheon, 417-840, Korea. 5College of Medi-cine, Seoul National University, Seoul, 110-744, Ko-rea. 6School of Medicine, Hanyang University, Seoul,471-701, Korea. 7College of Natural Science, SunchonNational University, Sunchon, 540-742, Korea. 8De-partment of Animal Science-Physiology, MichiganState University, East Lansing, MI 48824, USA.
*To whom correspondence should be addressed. E-mail: [email protected] (W.S.H.); [email protected] (S.Y.M.)
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(among others). Previous reports have de-scribed the generation of bovine ES-likecells (4) and mouse ES cells from the ICMsof cloned blastocysts (5–7) and the devel-opment of cloned human embryos to the 8-to 10-cell stage (8, 9). Here we describeevidence of the derivation of human EScells after SCNT (10).
Fresh oocytes and cumulus cells were do-nated by healthy women for the express pur-pose of SCNT stem cell derivation for ther-apeutic cloning research and its applications.Before beginning any experiments, we ob-tained approval for this study from the Insti-tutional Review Board on Human SubjectsResearch and Ethics Committees (HanyangUniversity Hospital, Seoul, Korea). Donorswere fully aware of the scope of our studyand signed an informed consent form (a sum-mary of the informed consent form is avail-able in the supporting online text); donorsvoluntarily donated oocytes and cumuluscells (including DNA) for therapeutic cloningresearch and its applications only, not forreproductive cloning; and there was no finan-cial payment. A total of 242 oocytes wereobtained from 16 volunteers (there were oneor two donors for each trial) after ovarianstimulation: 176 metaphase II (MII) oocyteswere used directly for SCNT, whereas theremaining 66 oocytes were allowed to matureto the MII stage before use in SCNT. Autol-ogous SCNT was performed; that is, the do-
nor’s own cumulus cell, isolated from thecumulus-oocyte complex (COC), was trans-ferred back into the donor’s own enucleatedoocyte. Before enucleation, the oocytes werematured in vitro in G1.2 medium (Vitro Life,Goteborg, Sweden) for 1 to 2 hours. Enucle-ation, SCNT, and electrical fusion were per-formed as described (11). To directly confirmthat the oocyte’s DNA was removed duringenucleation, we imaged the extruded DNAMII spindle complex from every oocyte withHoechst 33342 fluorescent DNA dye (Fig. 1,A and B; arrows).
Without any report of an efficient protocolfor human SCNT, several critical steps had tobe optimized (2), including reprogrammingtime, activation method, and in vitro cultureconditions. Reprogramming time, or the lapseof time between cell fusion and egg activa-tion, returns the gene expression of the so-matic cell to that needed for appropriateembryonic development. Initially, we inves-tigated the effect of simultaneous fusion andactivation, as used for porcine SCNT (12,13), but observed low fusion and cleavagerates, with no blastocyst development. In-stead, we adapted the bovine SCNT proce-dure of waiting a few hours between fusionand activation. By allowing 2 hours for re-programming, we were able to develop�25% of the embryos to the blastocyst stage.
Because sperm-mediated activation is absentin SCNT, an artificial stimulus is needed to
initiate development. Various chemical, physi-cal, and mechanical agents induce parthenoge-netic development in mice (14), but human dataare limited. Oocyte activation using the calciumionophore A23187 (calcimycin) or ionomycinand the protein synthesis inhibitor puromycininduces parthenogenetic development of humanoocytes at different efficiencies (15). We foundthat incubation in 10 �M A23187 for 5 min,followed by incubation with 2.0 mM 6-dimeth-ylaminopurine (DMAP) for 4 hours, gave effi-cient chemical activation for human SCNT eggs.Other investigators have reported encouragingresults in overcoming inefficiencies in embryoculture by supplementing the culture with differ-ent energy substrates (16). Furthermore, the re-cent development of serum-free sequential me-dia has led to considerable improvement in therate of clinical pregnancies produced by in vitrofertilization (IVF) (17). In this study, humanmodified synthetic oviductal fluid with aminoacids (hmSOFaa) was prepared by supplement-ing mSOFaa (18) with human serum albumin(10 mg/ml) and fructose (1.5 mM) instead ofbovine serum albumin (8 mg/ml) and glucose(1.5 mM). The replacement of glucose with fruc-tose improves the developmental competence ofbovine SCNT embryos (11, 19). Culture of hu-man SCNT-derived embryos in G1.2 mediumfor the first 48 hours followed by hmSOFaamedium produced more blastocysts, as com-pared to culture in G1.2 medium for the first 48hours followed by culture in G1.2 medium or incontinuous hmSOFaa medium (Table 1). Cibelliet al. (8) reported that the treatment of humanoocytes with 5 �M calcium ionomycin followedby 2 mM DMAP in G1.2 culture medium trig-gered pronucleus formation, embryonic cleav-age, and the formation of a blastocoelic cavity inhuman parthenotes. However, they did not ob-tain human SCNT blastocysts when their proto-col was applied to SCNT embryos. Limitationsin oocyte supply precluded full optimization ofall the parameters for human SCNT; nonethe-less, the protocol described here producedcloned blastocysts at rates of 19 to 29% (as apercentage of oocytes used) and was comparableto those produced by established SCNT methodsin cattle (�25%) (11) and pigs (�26%) (12, 13).
A total of 30 SCNT-derived blastocysts werecultured, 20 ICMs were isolated by immunosur-gical removal of the trophoblast, and one ES cellline (SCNT-hES-1) was derived. The resultingSCNT-hES-1 cells had a high nucleus-to-cyto-plasm ratio and prominent nucleoli. The cellcolonies displayed similar morphology to thatreported previously for hES cells derived afterIVF (Fig. 1, C to E). When cultured in definedmedium conditioned for neural cell differentia-tion (20), SCNT-hES-1 cells differentiated intonestin-positive cells, an indication of primitiveneuroectoderm differentiation (Fig. 1F). TheSCNT-hES-1 cell line was mechanically pas-saged by dissociation every 5 to 7 days andsuccessfully maintained its undifferentiated mor-
Fig. 1. Confirmation of enucleation, photographs of human SCNT ES cells and their differentiatedprogeny, and karyotype analysis. (A and B) Images (�200) of extruded DNA MII spindle complexes(arrows) from an oocyte before (A) and after (B) enucleation. (C to E) Bright-field [(C), �100] andphase contrast [(D), �100] micrographs and higher magnification image [(E), �200] of a colony ofSCNT-hES-1 cells. Immunofluorescence staining for nestin [(F), �200] and G-banded kayotyping(G) in SCNT-hES-1 cells are shown. Scale bars, 20 �m in (A) and (B) and 100 �m in (C) to (F).
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phology after continuous proliferation for �70passages, while still maintaining a normal fe-male (XX) karyotype (Fig. 1G) (21). Further-more, the SCNT-hES-1 cells expressed ES cellmarkers such as alkaline phosphatase, SSEA-3,SSEA-4, TRA-1-60, TRA-1-81, and Oct-4, butnot SSEA-1 (Fig. 2). As previously described inmonkey (22) and human ES cells (1, 23, 24) andin mouse SCNT-ES cells (6), SCNT-hES-1 cellsdid not respond to exogenous leukaemia inhibi-tory factor, suggesting that a pluripotent state ismaintained by a gp130-independent pathway.The pluripotency of SCNT-hES-1 cells was in-vestigated in vitro (fig. S1) and in vivo (Fig. 3).Clumps of the cells were cultured in vitro insuspension to form embryoid bodies. The result-ing embryoid bodies were stained with threedermal markers and were found to differentiateinto a variety of cell types, including derivativesof endoderm, mesoderm, and ectoderm (fig. S1).When undifferentiated SCNT-hES-1 cells wereinjected into the testes of severe combined im-munodeficient (SCID) mice, teratomas were ob-tained 6 to 7 weeks after injection. The resultingteratomas contained tissue representative of allthree germ layers. Differentiated tissues seen inFig. 3 include neuroepithelial rosset, pigmentedretinal epithelium, smooth muscle, bone, car-tilage, connective tissues, and glandular ep-ithelium. The DNA fingerprinting analysiswith human short tandem repeat (STR)markers indicates that the cell line originat-ed from the cloned blastocysts reconstructedfrom the donor cells, not from parthenoge-netic activation (Fig. 4, A to C). The statis-tical probability that the cells may havederived from an unrelated donor is 8.8 �10–16. Reverse transcription polymerasechain reaction (RT-PCR) amplification forpaternally expressed (hSNRPN and ARH1)and maternally expressed (UBE3A and H19)genes further confirmed that the cell lineoriginated from the donor cells (Fig. 4D).
Simerly et al. (26) recently reporteddefective mitotic spindles after SCNT innonhuman primate embryos, perhaps re-sulting from the depletion of microtubulemotor and centrosome proteins lost to themeiotic spindle after enucleation. In thisstudy, Fig. 1G demonstrates that SCNT-hES-1 cells have the normal karyotype. Wespeculate that SCNT blastocysts fromwhich ES cell lines were not derived mighthave been aneuploid. However, it is impor-tant to note that our investigations differ fromthose of Simerly et al. in a few ways: Mediaand protocols for in vitro development wereoptimized for human oocytes and embryos,whereas the protocols for nonhuman primatestudies are extrapolated from clinical proce-dures; the enucleation method here differs, be-cause we squeeze the MII oocyte so that theDNA spindle complex is extruded through asmall hole in the zona pellucida, instead ofaspirating the DNA spindle complex with a
glass pipette as others have described (27); andthe DNA spindle complex is extruded shortlyafter the appearance of the first polar body, sothat it may even be at the prometaphase II stage.
In this report, we provide three lines of
evidence supporting the nuclear transfer ori-gins of the SCNT-hES-1 cell line: (i) DNAextraction was verified for each of the 242enucleated oocytes (Fig. 1, A and B; arrows);(ii) DNA fingerprinting showed heterozy-
Fig. 2. Expression of characteristic cell surface markers in human SCNT ES cells. SCNT-hES-1cells expressed cell surface markers, including alkaline phosphatase (B), SSEA-3 (H), SSEA-4 (K),TRA-1-60 (N), TRA-1-81 (Q), and Oct-4 (T), but not SSEA-1 (E). The differentiated SCNT-hES-1cells were not stained with alkaline phosphatase (A). The IVF-derived human ES cells (Miz-hES)were used for comparison and also expressed alkaline phosphatase (C), SSEA-3 (I), SSEA-4 (L),TRA-1-60 (O), TRA-1-81 (R), and Oct-4 (U), but not SSEA-1 (F). Negative controls not treatedwith first antibodies are shown (D, G, J, M, P, and S). Magnification in (A) to (U), �40. Scalebars, 100 �m.
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gous, not homozygous, chromosomes (Fig. 4,A to C); and (iii) RT-PCR showed biparental,and not unimaternal, expression of imprintedgenes (Fig. 4D). Although the Cyno-1 parthe-nogenetic cells retained their strictly maternalimprints, that evidence came from a singlemonkey cell line. Given the aberrant expres-sion of imprinted genes after murine SCNT(28), perhaps the SCNT-hES-1 cells’ bipa-rental expression of imprinted genes mighthave been influenced by SCNT or subsequentculture. Heterologous along with autologousSCNT will provide more definitive molecularevidence. Although overwhelming ethicalconstraints preclude any reproductive cloningattempts, complementary investigations innonhuman primates might provide additionaland confirmatory information. Consequently,although we cannot exclude the possibility ofa parthenogenetic origin, the studies reportedhere support the conclusion that the SCNT-hES-1 cell line originated from the donor’sdiploid somatic cumulus cell after SCNT.
In order to successfully derive immuno-compatible human ES cells from a livingdonor, a reliable and efficient method forproducing cloned embryos and ES isolationmust be developed. Thomson et al. (1),Reubinoff et al. (23), and Lanzendorf et al.(29) produced human ES cell lines at highefficiency. Briefly, five ES cell lines werederived from a total of 14 ICMs, two EScell lines were derived from four ICMs, andthree ES cell lines were derived from 18ICMS, respectively. In our study, one SCNT-hES cell line was derived from 20 ICMs. It
remains to be determined whether this low ef-ficiency is due to faulty reprogramming of thesomatic cells or to subtle variations in our ex-perimental procedures. We cannot rule out the
possibility that the genetic background of thecell donor had an impact on the overall effi-ciency of the procedure. Further improve-ments in SCNT protocols and in vitro culture
Fig. 3. Teratomas formedby human SCNT ES cellsin the testes of SCIDmiceat 12 weeks after injec-tion. Neuroepithelial ros-set (A), pigmented retinalepithelium (B), ostoid is-land showing bony differ-entiation (C), cartilage(D), and glandular epithe-liumwith smoothmuscleand connective tissues(E). Magnification in (A)to (D), �200; in (E),�100. Scale bar, 100�m.
Table 1. Conditions for human SCNT.
Experiment Activation condition*Reprogrammingtime (hours)
1st stepmedium†
2nd stepmedium
No. ofoocytes
No. (%) of cloned embryos developed to
Two-cell Compacted morula Blastocyst
1st set 10 �Mionophore
6-DMAP 2 G 1.2 hmSOFaa 16 16 (100) 4 (25) 4 (25)
10 �Mionophore
6-DMAP 4 G 1.2 hmSOFaa 16 15 (94) 1 (6) 0
10 �Mionophore
6-DMAP 6 G 1.2 hmSOFaa 16 15 (94) 1 (6) 1 (6)
10 �Mionophore
6-DMAP 20 G 1.2 hmSOFaa 16 9 (56) 1 (6) 0
2nd set 10 �Mionophore
6-DMAP 2 G 1.2 hmSOFaa 16 16 (100) 5 (31) 3 (19)
5 �Mionophore
6-DMAP 2 G 1.2 hmSOFaa 16 11 (69) 0 0
10 �Mionomycin
6-DMAP 2 G 1.2 hmSOFaa 16 12 (75) 0 0
5 �Mionomycin
6-DMAP 2 G 1.2 hmSOFaa 16 9 (56) 0 0
3rd set 10 �Mionophore
6-DMAP 2 G 1.2 hmSOFaa 16 16 (100) 4 (25) 3 (19)
10 �Mionophore
6-DMAP 2 G 1.2 G 2.2 16 16 (100) 0 0
10 �Mionophore
6-DMAP 2 Continuous hmSOFaa 16 16 (100) 0 0
4th set 10 �Mionophore
6-DMAP 2 G 1.2 hmSOFaa 66 62 (93) 24 (36) 19 (29)
*Fused donor oocytes and somatic cells were activated in either calcium ionophore A23187 (5 or 10 �M) or ionomycin (5 or 10 �M) for 5 min, followed by 2 mM 6-DMAP treatmentfor 4 hours. †Oocytes were incubated in the first medium for 48 hours.
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A C
DBFig. 4. DNA finger-printing analysis and ex-pression of imprintedgenes. (A) Isogenic anal-ysis in loci D3S1358(chromosome location3p), vWA (chromosomelocation 12p12-pter),and FGA (chromosomelocation 4q28). (B) Iso-genic analysis in lociamelogenin (chromo-some location X:p22.1-22.3 and Y:p11.2),THO1 (chromosome lo-cation 11p15.5), TPOX
(chromosome location 2p23-2per), and CSF1PO (chromosome location5q33.3-34). (C) Isogenic analysis in loci D5S818 (chromosome location5p22-31), D13S317 (chromosome location 13q22-31), and D7S820(chromosome location 7q11.21-22). The boxed numbers and corre-sponding peaks represent locations of polymorphisms for each shorttandem repeat marker. (D) RT-PCR amplification of paternally ex-pressed (hSNRPN and ARH1) and maternally expressed (UBE3A andH19) genes. Cyno-1, maternally derived monkey parthenogenetic stemcell line (25); mFBLST, monkey fibroblasts; hFBLST, human fibroblasts;GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Tm(-), withouttemplate for PCR amplification.
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systems are needed before contemplating theuse of this technique for cell therapy. Inaddition, the mechanisms governing the dif-ferentiation of human tissues must be eluci-dated in order to produce tissue-specific cellpopulations from undifferentiated ES cells.This study shows the feasibility of generatinghuman ES cells from a somatic cell isolatedfrom a living person.
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togenetic Nomenclature (S. Karger, Basel, Switzer-land, 1995).
22. J. A. Thomson et al., Proc. Natl. Acad. Sci. U.S.A. 92,7844 (1995).
23. B. E. Reubinoff, F. P. Pera, C.-Y. Fong, A. Trounson, A.Bongso, Nature Biotechnol. 18, 399 (2000).
24. S. R. John, Trends Biotechnol. 20, 417 (2002).25. K. E. Vrana et al., Proc. Natl. Acad. Sci. U.S.A. 100
(suppl. 1), 1911 (2003).26. C. Simerly et al., Science, 300, 297 (2003).27. I. Wilmut et al., Nature 385, 810 (1997).28. D. Humphreys et al., Proc. Natl. Acad. Sci. U.S.A. 99,
12889 (2002).29. S. E. Lanzendorf et al., Fertil. Steril. 76, 132 (2001).30. We thank Y. Y Hwang (Hanyang University) for as-
sistance with oocyte collections; S. I. Rho (MizMedi
Hospital), H. S. Yoon (MizMedi Hospital,) and S. K. Oh(Seoul National University) for assistance on hES cellsculture; Y. K. Choi (Korea Research Institute of Bio-science and Biotechnology) for assistance on terato-ma formation; Tak Ko (Michigan State University) forgene expression analysis of Cyno-1 cells; and A.Trounson (Monash University), B. D. Bavister (Univer-sity of New Orleans), and D. P. Wolf (Oregon Na-tional Primate Research Center) for critical review ofthe manuscript. J. B. Cibelli made intellectual contri-butions to the manuscript and the RNA analysis ofnonhuman primate cells. All human experimentswere performed in Korea by Korean scientists. Thisstudy was supported by grants from Advanced Back-bone IT Technology Development (grant IMT2000-C1-1) to W.S.H. and the Stem Cell Research Center(grant M102KL0100-02K1201-00223) to S.Y.M. Theauthors are grateful for a graduate fellowship provid-ed by the Ministry of Education through the BK21program.
Supporting Online Materialwww.sciencemag.org/cgi/content/full/1094515/DC1Materials and MethodsSOM TextFig. S1
9 December 2003; accepted 4 February 2004Published online 12 February 2004;10.1126/science.1094515Include this information when citing this paper.
Force-Clamp SpectroscopyMonitors the Folding Trajectory
of a Single ProteinJulio M. Fernandez* and Hongbin Li
Weused force-clamp atomic forcemicoscopy tomeasure the end-to-end lengthof the small protein ubiquitin during its folding reaction at the single-moleculelevel. Ubiquitin was first unfolded and extended at a high force, then thestretching force was quenched and protein folding was observed. The foldingtrajectories were continuous and marked by several distinct stages. The timetaken to fold was dependent on the contour length of the unfolded protein andthe stretching force applied during folding. The folding collapse was marked bylarge fluctuations in the end-to-end length of the protein, but these fluctuationsvanished upon the final folding contraction. These direct observations of thecomplete folding trajectory of a protein provide a benchmark to determine thephysical basis of the folding reaction.
Resolving the folding pathway of a proteinremains a challenge in biology (1–9). Here,we demonstrate a method by which the entirefolding trajectory of a single protein can berecorded as a function of time. We usedsingle-molecule atomic force micoscopytechniques (10, 11) in the force-clamp mode(12, 13) to apply a constant force to a singlepolyprotein composed of nine repeats of thesmall protein ubiquitin (13–16). This resultedin the probabilistic unfolding of ubiquitin,which was observed as stepwise elongationsof the protein in which each step correspond-
ed to the unfolding of an individual proteinmodule (12). We applied this technique tomonitor the end-to-end length of a singleubiquitin polyprotein (17) during reversibleunfolding-folding cycles. Our experimentalapproach is illustrated in Fig. 1. Figure 1Ashows the changes in the length of a singleubiquitin polyprotein in response to thestretching force displayed in Fig. 1B. Asshown, stretching the polyubiquitin chain at120 pN triggers a series of unfolding eventsthat appear as a staircase of 20-nm steps,marking the unfolding of the individual ubiq-uitins in the chain (Fig. 1A). After 4 s, theforce was relaxed to 15 pN (Fig. 1B) (18),and we observed the protein spontaneouslycontract in stages until it reached its foldedlength (Fig. 1A). To confirm that the polypro-
tein had folded, we raised the stretching forceback to 120 pN at 14 s (Fig. 1B) and observedthe ubiquitin chain extend in steps of 20 nmback to its fully unfolded length (Fig. 1A).Hence, the spontaneous contraction of theprotein observed upon reducing the forcefrom 120 pN down to 15 pN corresponds tothe folding trajectory of the mechanicallyunfolded ubiquitin.
We observed and analyzed 81 foldingevents similar to those shown in Fig. 1. Twotypical folding trajectories for mechanicallyunfolded polyubiquitin molecules are shownin Fig. 2. Most of the folding trajectories arequalitatively similar, following a continuousconvex time course marked by abrupt chang-es in slope. However, we have never ob-served identical sets of trajectories, indicatingthe existence of multiple folding pathwaysfor ubiquitin. To simplify the analysis of thefolding trajectories, we divided their timecourse, roughly, into four distinct stagesmarked by abrupt changes in the slope of thecollapse (Fig. 2). As an example, we analyzethe recording shown in Fig. 2A. The firststage (1 in Fig. 2A and inset) is fast, lasting�10 ms, which is slower than the time ittakes the force to reach its set point (�3 ms inthis experiment). The collapse rate for thisstage (cr1 � 2135 nm/s) is within the rangebut clearly slower than the maximum rate ofchange, or slew rate (sr), of the feedbackduring this experiment (measured at sr ��8300 nm/s, after the molecule detachedfrom the cantilever). This stage is likely tocorrespond to the elastic recoil of the unfold-ed polypeptide chain adjusting its length tothe step change in the pulling force. Thisstage is always fast and is clearly marked in
Department of Biological Sciences, Columbia Univer-sity, New York, NY 10027, USA.
*To whom correspondence should be addressed. E-mail: [email protected]
R E P O R T S
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1www.sciencemag.org SCIENCE Erratum post date 16 DECEMBER 2005
post date 16 December 2005
ERRATUM
C O R R E C T I O N S A N D C L A R I F I C A T I O N S
Reports: “Evidence of a pluripotent human embryonic stem cell line derived
from a cloned blastocyst” by W. S. Hwang et al. (12 Mar. 2004, p. 1669). Con-
trary to the statements in the second paragraph of text and first paragraph of
the supporting online material, which indicated that there was no financial pay-
ment to oocyte and cumulus cell donors, some oocyte donors were financially
compensated for their donation with a payment of approximately U.S. $1,400.
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RETRACTION
www.sciencemag.org SCIENCE RETRACTION POST DATE 12 JANUARY 2006 1
CORRECTIONS &CLARIFICATIONS
The final report from the Investigation Committee of Seoul National University (SNU) (1) has
concluded that the authors of two papers published in Science (2, 3) have engaged in research
misconduct and that the papers contain fabricated data. With regard to Hwang et al., 2004 (2),
the Investigation Committee reported that the data showing that DNA from human embryonic
stem cell line NT-1 is identical to that of the donor are invalid because they are the result of
fabrication, as is the evidence that NT-1 is a bona fide stem cell line. Further, the committee
found that the claim in Hwang et al., 2005 (3) that 11 patient-specific embryonic stem cells
line were derived from cloned blastocysts is based on fabricated data. According to the report
of the Investigation Committee, the laboratory “does not possess patient-specific stem cell lines
or any scientific basis for claiming to have created one.” Because the final report of the SNU
investigation indicated that a significant amount of the data presented in both papers is fabri-
cated, the editors of Science feel that an immediate and unconditional retraction of both
papers is needed. We therefore retract these two papers and advise the scientific community
that the results reported in them are deemed to be invalid.
As we post this retraction, seven of the 15 authors of Hwang et al., 2004 (2) have agreed
to retract their paper. All of the authors of Hwang et al., 2005 (3) have agreed to retract
their paper.
Science regrets the time that the peer reviewers and others spent evaluating these papers
as well as the time and resources that the scientific community may have spent trying to
replicate these results.Donald Kennedy
Editor-in-Chief
References1. Investigation Committee Report, Seoul National University, 10 Jan. 2006. (Members: Chairman Myung-Hee Chung, SNU,
Uhtaek Oh, SNU, Hong-Hee Kim, SNU, Un Jong Pak, SNU, Yong Sung Lee, Hanyang University, In Won Lee, SNU, In Kwon Chung,
Yonsei University, Jin Ho Chung, SNU).
2. W.-S. Hwang et al., Evidence of A Pluripotent Human Embryonic Stem Cell Line Derived From a Cloned Blastocyst, Science 303,
1669 (2004).
3. W.-S. Hwang et al., Patient-Specific Embryonic Stem Cells Derived from Human SCNT Blastocysts, Science 308, 1777 (2005).
Published online 12 January 2006; in print 20 January 2006
10.1126/science.1124926
Post date 12 January 2006
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Asian Scientists and
the “Glass Ceiling”
IN HIS ARTICLE “A GLASS CEILING FOR ASIAN
scientists?” (News Focus, 28 Oct., p. 606),J. Mervis raised the interesting question ofwhy Asian scientists, who are abundantlypresent in U.S. research and industrial labsand academic institutes, do not hold posi-tions and titles as seniorleaders. This is stronglysupported by available sta-tistics from the NationalInstitutes of Health, theSociety for Neuroscience,and the American Societyfor Biology and MolecularBiology.
We applaud these effortsin revealing some of thefacts and concerns behindthis “glass ceiling.” Fromwhat we have gathered andwhat has been shown, Asian-Americans are representedcurrently in less than 10% ofhigher decision-makingpositions in their particularprofessions. This is true inacademia, government, and private enter-prise. Although the reasons behind thisissue are complicated and certainly need tobe dealt with appropriately, we believe it isand will continue to be America’s loss fornot tapping into such a vastly talented poolfor current and future leaders. It is alsotime for Asian-American scientists toassert themselves and to embrace any chal-lenges from the decision-making hierarchy.Our hope remains that Asian-Americansand non–Asian-Americans will worktogether to remove barriers for the commongood of this great nation. On this note, it isencouraging to see that some of the majorinstitutions in this country have appointedAsian-Americans to prominent leadershippositions; for example, two of the seven chairsin the Basic Science Research Departments atthe M.D. Anderson Cancer Center are held byAsian-Americans.
MIEN-CHIE HUNG,1 KENNETH FONG,2
JOSEPH K.-K. LI3*
1President of the Society of Chinese Bioscientists in
America (SCBA) and Professor and Chair, Depart-
ment of Molecular and Cellular Oncology,
University of Texas M.D. Anderson Cancer Center,
Houston, TX 77030, USA. 2President-elect of the
SCBA and Chairman, Kenson Venture, LLC, Palo Alto,
CA 94301, USA. 3Executive Director of the SCBA
and Professor, Department of Biology, Utah State
University, Logan, UT 84322–5305, USA.
*To whom correspondence should be addressed.
E-mail: [email protected]
WE READ THE NEWS FOCUS ARTICLE “A GLASS
ceiling for Asian scientists?” (J. Mervis, 28Oct., p. 606) with chagrin and numbers envy.Latino/Hispanic scientists at the NationalInstitutes of Health (NIH) are essentially anendangered species, despite a 10-year effort
by Hispanic scientists to havethe NIH address the disparity(see http://heo.nih.gov/). Incontrast to Asian scientists, inApril 2005 Latinos made up5.3% of tenure-track investiga-tors versus 4.5% in 1994(21.5% and 10.2%, respec-tively, for Asians) and 2.5% ofsenior investigators (1.5% in1994) (9.2% and 7.2%, respec-tively, for Asians) (1). In fact,Latinos account for 4.3% ofmedical officers, 3.4% of biol-ogists, and 2.5% of chemists inthe NIH workforce—signifi-cantly below the figure for theU.S. Civilian Labor Force (2).Hence, there seems to be littleopportunity for Latinos to
become the “right-hand men” (or women)noted by Dr. Jeang in the article.
RAYMOND MEJIA,* OFELIA OLIVERO,† MIGDALIA
RIVERA-GOBA,‡ ANA ANDERS,* CARLOS CABAN,*
MARTA LEON-MONZON,* ERNEST MARQUEZ*
National Institutes of Health, Bethesda, MD 20892,
USA.
*Past presidents of the NIH Hispanic Employee
Organization (NIH-HEO).
†Current president of the NIH-HEO.
‡President-elect of the NIH-HEO.
References1. See http://heo.nih.gov/files/Tenure_Track_and_
Tenured_Scientists.ppt.2. See http://heo.nih.gov/files/NIH_PROFILE_BY_JOB_
SERIES.ppt.
THE NEWS FOCUS ARTICLE “A GLASS CEILING
for Asian scientists?” by J. Mervis (28Oct., p. 606) attracted my attention. Whenyou look at these numbers, on the surface,one may draw the conclusion that Asianscientists are underrepresented. However,it should be taken into consideration thatmany Chinese scientists have been in theUnited States for less than 20 years, sinceChina’s “Open Door” policy. With gradu-ate study, postdoctoral training, and juniorfaculty positions, a scientist may needmore than 15 years to build up her or hisexperience and reputation to be a leader inresearch laboratories and in scientif ic
societies. The visibility of Asian scientistsin scientif ic leadership should increasewith time.
DIANHUA JIANG
Yale University School of Medicine, 333 Cedar
Street, New Haven, CT 06520–8057, USA. E-mail:
I WAS APPALLED WHEN I READ ABOUT THE
percentage of Asians in tenure-track posi-tions at the National Institutes of Health(NIH) in the News Focus article “A glassceiling for Asian scientists?” (J. Mervis, 28Oct., p. 606). According to the 2000 Census,Asians make up only 3.6% of the popula-tion of the United States, yet 21.5% ofresearchers at NIH are Asian. Could this bedue to a hiring bias on the part of NIH? I’mnot Asian so apparently I do not have theinherent qualities to make a good researcherand have had to forge out on my own to con-duct science. Bigotry at workplaces likeNIH will only harm our country in the longterm, and I hope that articles like this, ratherthan postulating some fanciful glass ceiling,will shed light on the immoral and unfairhiring practices that go on currently at ourpublic institutions.
JEFFREY B. STEWART
Aeri Park Institute of Molecular Biology and its
Applications (APIMBA), 3000 Kent Avenue, West
Lafayette, IN 47906, USA.
I WRITE TO ADDRESS COMMENTS ATTRIB-uted to me in the News Focus article “Aglass ceiling for Asian scientists?” (J.Mervis, 28 Oct., p. 606). Some commentscited in the article were from an e-mail andreferred to trainees and the diff icultiesthey, particularly those who are foreignnationals, face in making the transition topositions as independent scientists. Forexample, my comments indicated thattrainees were “not in a position” to serveon the committees of scientific societiesbecause they are at a very early stage oftheir careers. I have spent the better part ofmy academic career working to increasethe representation of women scientists atsenior levels. Having actively advocatedfor equity in the promotion and participa-tion of women scientists, I am hardly infavor of bias against other groups.
Scientific societies are not involved inthe hiring or promotion of their membersand thus do not control the numbers ofunderrepresented minorities who advanceto the faculty ranks from which societycommittee members are drawn. Scientificsocieties can, however, ensure that theaccomplishments of all their members are
LETTERS
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recognized through presentations at soci-ety-sponsored meetings as well as serviceon society committees. They can also take aleadership role in ensuring that the issue ofequity remains a priority within the scien-tific community as a whole. I believe thatthis article has raised the awareness of all ofus to this problem and hope that it will helpto achieve equity for all members of the sci-entific community.
LINDA J. PIKE
Department of Biochemistry and Molecular
Biophysics, Washington University School of
Medicine, 660 South Euclid, Box 8231, St. Louis, MO
63110, USA.
How to Cut World
Hunger in Half
THE CO-CHAIRS OF THE UN TASK FORCE ON
Hunger, created in 2002 to determine howto meet the UN hunger MillenniumDevelopment Goal (1, 2), recently summa-rized their f indings in Science (“Cuttingworld hunger in half,” P. A. Sanchez andM. S. Swaminathan, Policy Forum, 21Jan., p. 357).
Roughly 80% of the world’s hungry livein rural areas of the least developedcountries and have nopower in society. Thesepeople are mainly farm-ers and need incentivesto invest in their land tointensify agriculturalproduction (3, 4). How-ever, urban, mostly well-educated people, who donot depend on agricul-ture for their survival,frequently determineagricultural policies (5–7).
The Task Force on Hunger report doesnot make its analysis from the interests ofthe poorest, rural people, nor does it assesshow farmers would get necessary incen-tives (such as better prices) for their agri-cultural products (8, 9). According to theUN Development Programme’s HumanDevelopment Report, “The basic problemto be addressed in the WTO [World TradeOrganization] negotiations on agriculturecan be summarized in three words: richcountry subsidies” [(10), p. 10]. Wealthycountries give 1 billion U.S. dollars peryear in agricultural aid to developing coun-tries, while they subsidize their own agri-culture with nearly 1 billion U.S. dollarsper day [(10), p. 130].
According to the Human DevelopmentReport “Rich country consumers and tax-payers are locked into financing policiesthat are destroying livelihoods in some ofthe world’s poorest countries… WTO rules
threaten to systematically reinforce thedisadvantages faced by developing coun-tries and to further skew the benefits ofglobal integration towards developedcountries… The unbalanced agenda pur-sued by rich countries and failure to tackleagricultural subsidies are at the core of theproblem” [(10), p. 10]. UN SecretaryGeneral Kof i Annan has said that “The[Millennium Development Goals] can bemet by 2015—but only if all involvedbreak with business as usual and dramati-cally accelerate and scale up action now”[(10), p. 5].
To avoid that disaster, wealthy coun-tries must accept free trade principles. Atthe WTO meeting in Hong Kong inDecember 2005, they must agree to (i)make deep cuts in wealthy countries’ sup-port for domestic agriculture and prohibitexport subsidies and (ii) make deep cuts inbarriers to exports from developing coun-tries (10).
According to Kirkpatrick and George,trade liberalization “can contribute posi-tively to the [Millennium Development]goal of eradicating extreme poverty andhunger” [(11), p. 3]. Hoekman et al. writethat a 50% reduction “in border protectionwill have a much larger positive impact on
developing economies’ exports and welfarethan a 50% reduction in agricultural subsi-dies” [(12), p. 175]. Price incentivesthrough cancelled trade barriers and subsi-dies (13, 14), as well as a redressed rural-urban balance in living conditions and pro-duction (7), are therefore necessary but notsufficient to halve the number of hungry by2015. In contrast to the effects of aid anddebt cancellation, the money from higheragricultural prices goes directly into thepockets of the poorest people and of thefarmers in poor countries.
PER LINDSKOG
Department of Science and Technology, Linköping
University, S-601 74 Norrköping, Sweden. E-mail:
1. UN Millennium Project, Investing in Development: APractical Plan to Achieve the Millennium DevelopmentGoals: Overview (Earthscan, London and Sterling,VA,2005) (available at www.unmillenniumproject.org/documents/MainReportComplete-lowres.pdf).
2. UN Millennium Project, Task Force on Hunger. HalvingHunger: It Can Be Done (Earthscan, London and
Sterling VA, 2005) (available at http://www.unmilleni-umproject.org/reports/tf_hunger.htm).
3. E. Boserup, The Conditions of Agricultural Growth: TheEconomics of Agrarian Change Under Population(Aldine, New York, 1965).
4. M. Tiffen, M. Mortimore, F. Gichuki, More People, LessErosion: Environmental Recovery in Kenya (Wiley,Chichester, 1994).
5. M. Lipton, Why Poor People Stay Poor: A Study of UrbanBias in World Development (Temple Smith, London,1977).
6. P. Lindskog, J. Lundqvist, Why Poor Children Stay Sick:The Human Ecology of Child Health and Welfare in RuralMalawi (Research Report no. 85, ScandinavianInstitute of African Studies, Uppsala, 1989).
7. P. D. Goldsmith, K. Gunjal, B. Ndarishikanye, Agric.Econ. 31, 33 (2004).
8. R.Thiele, Afr. Dev. Rev. Rev. Afr. Dev. 15, 425 (2003).9. D. Tilman, K.G. Cassman, P. A. Matson, R. Naylor, S.
Polasky, Nature 418, 671 (2002).10. United Nations Development Programme, Human
Development Report (UNDP, New York, 2005) (avail-able at http://hdr.undp.org/reports/global/2005/).
11. C . Kirkpatrick, C . George, Sustainability ImpactAssessment of Proposed WTO Negotiations (ImpactAssessment Research Centre, University ofManchester, 22 April 2005) (available at www.sia-trade.org/wto/FinalOverallApr05.pdf).
12. B. Hoekman, F. Ng, M. Olarreaga, World Bank Econ. Rev.18, 175 (2004).
13. K.Anderson,W. Martin, World Econ. 28, 1301 (2005).14. M. A. Aksoy, J. C. Beghin, Eds. Global Agricultural Trade
and Developing Countries (World Bank, Washington,DC, 2005).
Cognitive Unbinding
in Sleep and Anesthesia
I READ WITH GREAT INTEREST THE REPORT
“Breakdown of cortical effective connectiv-ity during sleep” by M. Massimini et al. (30Sept., p. 2228). As in the f ield of sleepresearch, the traditional view in anesthesi-ology has been that the brain is somehow“shut off ” under general anesthesia. Morerecent data and formulations of anestheticmechanism suggest, however, that it is notthe neural activity but rather the integrationof neural information that is inhibited.
It has been previously reported thatnumerous general anesthetic agents fromvarious pharmacologic classes induce asimilar pattern of functional uncouplingbetween the cerebral hemispheres, as wellas between the rostral and caudal poles ofthe brain (1). These and other data describ-ing anesthetic-induced interruption ofcognitive binding processes have givenrise to the theory of general anesthesia as a“cognitive unbinding” (2, 3). Recent datafrom magnetic resonance imaging supportthis by demonstrating loss of functionalcortical connectivity under sevofluraneanesthesia (4).
The integration of cognitive informa-tion as an essential feature of conscious-ness has its philosophical origins in the18th-century epistemology of Kant and hasmade its modern neuroscientific appear-ance as the cognitive binding problem andthe information integration theory ofTononi (5). Massimini et al. suggest that
“In contrast to the effects of aid
and debt cancellation,the moneyfrom higher agricultural prices
goes directly into the pockets of
the poorest people....”
16 DECEMBER 2005 VOL 310 SCIENCE www.sciencemag.org
L E T T E R S
–LINDSKOG
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the loss of integration during sleep sup-ports its central role in consciousness, aclaim that is strengthened by current per-spectives of the general anesthetic mecha-nism. Furthermore, I would suggest thatthe loss of cortical effective connectivity,or “cognitive unbinding,” may be the com-mon feature of general anesthesia andsleep that has long been hypothesized butnever identified.
GEORGE A. MASHOUR
Anesthesia and Critical Care, Massachusetts
General Hospital, Clinics 309, 55 Fruit Street,
Boston, MA 02138, USA.
References1. E. R. John et al., Conscious. Cogn. 10, 165 (2001).2. G.A. Mashour, Anesthesiology 100, 428 (2005).3. E. R. John, L. S. Prichep, Anesthesiology 102, 447
(2005).4. S. J. Peltier et al., Neuroreport 16, 285 (2005).5. G.Tononi, BMC Neurosci. 5, 42 (2004).
ResponseMASHOUR’S COMMENTS GO RIGHT TO THE
point. According to the information inte-gration theory (1), the neural substrate ofhuman consciousness is a complex of neu-ronal groups, largely contained within thethalamocortical system, that has two keyproperties: (i) it behaves as a single, inte-grated entity and (ii) it has a large repertoireof different states or firing patterns. Such acombination of properties requires that dis-tributed neuronal groups that are function-ally specialized can interact effectivelythrough multiple pathways.
If the critical interactions among suchspecialized neuronal groups are compro-mised, consciousness should fade, even ifneural activity is preserved. Our Reportindicates that this is precisely what hap-pens during slow wave sleep early in thenight. Mashour and others have pointed outthat a similar situation may occur underanesthesia (2–4). For example, recent neu-roimaging studies have revealed that func-tional connectivity (correlated activity)between distant cortical areas and betweenthalamus and cortex is reduced by differentanesthetic agents (5).
An important task for the future will beto determine whether effective connectivity(the ability of one cortical area to causally
affect other areas) is also reduced by anes-thetic action. It would be important to knowwhether a reduction of effective connectiv-ity also underlies the anesthetic action ofketamine and other substances that do notreduce cortical metabolism (4). It wouldalso be important to establish whether abreakdown in cortical effective connectiv-ity is due primarily to a direct action byanesthetics on cortico-cortical connections,or if it results indirectly from a depressionof cortico-thalamic and thalamo-corticalinteractions. Although many anestheticshave a disproportionate effect on thalamicmetabolism or blood flow (6), this may bean inevitable consequence of a diffusereduction in cortical input to the thalamus(4). Finally, it would be valuable to deter-
mine whether the transition between just-conscious to unconscious that often occurswith slight changes in anesthetic concentra-tion is predicted more reliably by changesin effective connectivity than by changes inoverall metabolism or neuronal activity (4).
GIULIO TONONI AND MARCELLO MASSIMINI
Department of Psychiatry, University of Wisconsin,
Madison, 6001 Research Park Boulevard, Madison,
WI 53719, USA.
References1. G.Tononi, BMC Neurosci. 5, 42 (2004).2. G.A. Mashour, Anesthesiology 100, 428 ( 2004).3. E. R. John, L. S. Prichep, Anesthesiology 102, 447
(2005).4. M. T. Alkire, J . Miller, Prog. Brain Res. 150 , 229
(2005).5. N. S.White, M.T.Alkire, Neuroimage 19, 402 (2003).6. M. T. Alkire, R. J. Haier, J. H. Fallon, Conscious Cogn. 9,
370 (2000).
Letters to the EditorLetters (~300 words) discuss material publishedin Science in the previous 6 months or issues ofgeneral interest. They can be submittedthrough the Web (www.submit2science.org) orby regular mail (1200 New York Ave., NW,Washington, DC 20005, USA). Letters are notacknowledged upon receipt, nor are authorsgenerally consulted before publication.Whether published in full or in part, letters aresubject to editing for clarity and space.
CORRECTIONS AND CLARIFICATIONS
Letters: “Response to ‘Problems of study-ing extinction risks’” by M. Cardillo et al.(25 Nov., p. 1277). In the third sentence ofthe last paragraph, “Despite the case forusing PICs in extinction risk studies havingrecently been clearly and elegantly made(6)…,” the reference citation is incorrect. Itshould be reference (7): D. O. Fisher, I. P. F.Owens, Trends Ecol. Evol. 19, 391 (2004).
Reports:“Patient-specific embryonic stemcells derived from human SCNT blasto-cysts” by W. S. Hwang et al. (17 June,p. 1777). There were errors in Table 2. Thecorrected table appears at right.
Reports : “Evidence of a pluripotenthuman embryonic stem cell line derivedfrom a cloned blastocyst” by W. S. Hwanget al. (12 Mar. 2004, p. 1669). Contrary tothe statements in the second paragraph oftext and first paragraph of the supportingonline material, which indicated that therewas no financial payment to oocyte andcumulus cell donors, some oocyte donorswere financially compensated for theirdonation with a payment of approxi-mately U.S. $1,400.
TECHNICAL COMMENT ABSTRACTS
COMMENT ON “Characterization of Excess Electrons in Water-ClusterAnions by Quantum Simulations”
J. R. R.Verlet,A. E. Bragg,A. Kammrath, O. Cheshnovsky, D. M. Neumark
The conclusion by Turi et al. (Reports, 5 August 2005, p. 914) that all experimental spectral and energetic data onwater-cluster anions point toward surface-bound electrons is overstated. Comparison of experimental verticaldetachment energies with their calculated values for (H2O)n
– clusters with surface-bound and internalized elec-trons supports previous arguments that both types of clusters exist.
Full text at www.sciencemag.org/cgi/content/full/310/5755/1769b
RESPONSE TO COMMENT ON “Characterization of Excess Electrons inWater-Cluster Anions by Quantum Simulations”
László Turi,Wen-Shyan Sheu, Peter J. Rossky
We reiterate that the conclusions of our original report are based on identifiable characteristic trends in severalobservables with cluster size.The numerical comparison between simulated and experimental vertical detach-ment energies emphasized by Verlet et al. reflects quantitative limitations of our atomistic model,but, in our opin-ion, does not undermine these conclusions.
Full text at www.sciencemag.org/cgi/content/full/310/5755/1769c
NT-hESC Isolation Plurip Differ Pass # DNA HLA
–2ZF -
BlastIdentical Match
–3 Blast � � Identical Match
–4, –5 Im mS � , EB Identical Match
–6, –7 Im mS � EB Identical Match
–8 Blast � EB Identical Match
–9ZF -
Blast�
�
EB Identical Match
–10 Im mS � EB Identical Match
–11ZF -
Blast� EB Identical Match
–12ZF -
BlastTBD TBD Identical Match
P40
P35
P26
P25
P21
P20
P19
P19
P7
� �
Table 2. Summary of patient-specific human NT-ESClines. ZF-blast, zona-free blasotcyst; ImmS, immuno-surgery; Plurip, Pluripotent; TBD, to be determined; EB,embryoid body; �, pluripotency demonstrated by bothEBs and teratomas. Normal karyotypes have been shownfor each line (female, pink; male, blue).
www.sciencemag.org SCIENCE VOL 310 16 DECEMBER 2005
L E T T E R S
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