Comment on "Molecular Correlates of Primate Nuclear Transfer Failures"
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We believe that Simerly et al. (1) have reported important new data but have overstated their conclusions. Their preliminary observations highlight the importance of egg quality—centrosome and spindle competence, specifically—in nuclear transfer (NT) procedures. However, the data are scant and their interpretation is compromised by the multiplicity of methods used. In our own experience, multiple factors including donor and recipient cell cycle stage and oocyte age can influence the integrity of the spindle complex. Simerly et al. buttressed their claim that primates are unique in the specific absence of NuMA (nuclear-mitotic apparatus protein) or HSET based only on immunofluorescence. However, one cannot conclude from these data that the oocyte was depleted of these proteins—only that they were not detected where expected using the techniques described. The occurrence of abnormal spindles has long plagued NT in most species, and few comparable immunocytochemical studies have been reported to support the conclusion that failed spindle complexes in other species would not lack those markers as well. More comprehensive investigations of NT-induced centrosome-spindle perturbations will reveal the true impact of these manipulations on the efficiency of NT on a species-byspecies basis.
Simerly et al. transferred only 33 rhesus embryos into 16 surrogates and concluded that reproductive cloning in primates may be unachievable. In a related news article (2), the senior author of (1) stated that it is almost as if someone "drew a sharp line between old-world primates—including people—and other animals, saying, `I'll let you clone cattle, mice, sheep, even rabbits and cats, but monkeys and humans require something more.'" In our own hands, it took dozens of embryos to generate Dolly (3), more than 150 embryos to generate the first cloned mouse pup (4), and 586 embryos to establish the first two pregnancies in pigs (5). Hundreds of other studies have ended with no pregnancies at all. We suggest that given the potential importance of nuclear transfer techniques in human cell therapies, conclusions regarding the efficiency of human NT extrapolated from animal data should be tempered with abundant caution.
Robert Lanza
Young Chung
Michael D. West
Advanced Cell Technology
One Innovation Drive
Worcester, MA 01605, USA
E-mail: [email protected]
Keith H. S. Campbell
Division of Biological Sciences
University of Nottingham
Loughborough, UK
References
1. C. Simerly et al., Science 300, 297 (2003).[Free Full Text]
2. G. Vogel, Science 300, 225 (2003).[Abstract/Free Full Text]
3. I. Wilmut, A. E. Schnleke, J. McWhir, A. J. Kind, K. H. S. Campbell, Nature 385, 810 (1997).[CrossRef][ISI][Medline]
4. Y. Chung, unpublished data.
5. I. A. Polejaeva et al., Nature 407, 86 (2000).[CrossRef][ISI][Medline]
17 April 2003; accepted 31 July 2003
10.1126/science.1085871
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Response to Comment on "Molecular Correlates of Primate Nuclear Transfer Failures"
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Space constraints precluded publication of bovine nuclear transfer (NT) data (Fig. 1) that may allay the concerns of Lanza et al (1). After primate NT, disorganized spindles with chaotically distributed chromosomes are always observed (2). Bovine NTs, however, organize a single microtubule aster emanating from the juxtanuclear NT centrosome at first interphase (Fig. 1A). Mitotic bovine NTs frequently assemble symmetrical bipolar spindles with precisely aligned chromosomes [Fig. 1B, blue and (2)], a situation never observed after primate NT. These and centrosome and kinesin NT data are subjects of a forthcoming report (3).
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Fig. 1. Chromosomes align on symmetrical mitotic spindles after bovine nuclear transfer, unlike the chaotic spindles detected after primate NT (4). (A) Somatic cell nuclear transfer in cattle oocytes results in a single radially arrayed microtubule aster at first interphase. (B) At first mitosis, bovine NTs organize accurate bipolar mitotic apparatus (green) with aligned chromosomes (blue). Scale bar: A, 10 µm; B, 5 µm. [View Larger Version of this Image (56K GIF file)]
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Without benefit of this additional knowledge, Lanza et al. fairly raise the uncertainty in interpreting NT results, especially in extrapolating assisted reproductive technology (ART) successes or failures between species. For example, >90% fertilization is routinely achieved using intracytoplasmic sperm injection (ICSI) in both humans and nonhuman primates (resulting in the global adoption of ICSI by ART clinics), whereas ICSI has produced few mice or cattle (4). Because NT successes vary, comparisons between Dolly's birth after 29 embryo transfers into 13 ewes (5) and our lack of established pregnancies after 33 rhesus embryo transfers are not reasonable. Primate ART, including NT, is uniquely challenging (6): Oocyte availability and quality, synchronized surrogates, cleavage-stage embryo transfers versus blastocysts transfers, and reproductive seasonality all present practical limitations. And unlike mice and domestic mammals, primates carry singletons. Finally, while the protocols for mouse and bovine NT are species-specific, primate laboratories must extrapolate from human ART clinics for simian-specific optimizations.
Although human embryonic stem cells (hESCs) derived after NT might overcome immune rejection, NT epigenetic abnormalities (7) and microchimerism in transplantation patients (8) encourage alternate strategies for tolerance or managing incompatibilities. In light of the available evidence, we maintain our original conclusion (2) that "[w]ith current approaches, [primate] NT to produce embryonic stem cells may prove difficult—and reproductive cloning unachievable." We agree with Lanza et al. (1) that interpretations regarding both NT and hESC data in primates, human (9, 10) and nonhuman (5, 11) alike, should be "tempered with abundant caution."
G. Schatten
C. Navara
C. Payne
S. Capuano
G. Gosman
K.-Y. Chong
D. Takahashi
C. Chace
L. Hewitson
C. Simerly
Pittsburgh Development Center,
Magee-Womens Research Institute,
Department of Obstetrics-Gynecology-
Reproductive Sciences and Cell
Biology-Physiology,
University of Pittsburgh School of Medicine
Pittsburgh, PA 15213, USA
E-mail: [email protected]
D. Compton
Department of Biochemistry
Dartmouth Medical School
Hanover, NH 03755, USA
T. Dominko
CellThera
Worcester, MA 01605, USA
References
1. R. Lanza, Y. Chung, M. D. West, K. H. S. Campbell, Science 301, 1482 (2003); www.sciencemag.org/cgi/content/full/301/5639/1482b.
2. C. Simerly et al., Science 300, 297 (2003).[Free Full Text]
3. C. Navara et al., unpublished results (2003).
4. G. Schatten, L. Hewitson, C. Simerly, P. Sutovsky, G. Huszar, J. Law Med. Ethics 26, 29 (1998).[ISI][Medline]
5. I. Wilmut, A. E. Schnleke, J. McWhir, A. J. Kind, K. H. S. Campbell, Nature 385, 810 (1997).[CrossRef][ISI][Medline]
6. L. Hewitson et al., Nature Med. 5, 431 (1999).[CrossRef][ISI][Medline]
7. K. Hochedlinger, R. Jaenisch, N. Engl. J. Med. 349, 275 (2003).[Free Full Text]
8. T. E. Starzl et al., Lancet 361, 1502 (2003).[CrossRef][ISI][Medline]
9. J. B. Cibelli et al., E-biomed J. Regener. Med. 2, 25 (2001).[CrossRef]
10. R. P. Lanza, J. B. Cibelli, M. D. West, Nature Biotechnol. 20, 117 (2002).[CrossRef][ISI][Medline]
11. J. B. Cibelli et al., Science 295, 819 (2002).[Free Full Text]
9 June 2003; accepted 8 August 2003
10.1126/science.1087756
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