Somatic cell nuclear transfer in its first and second decades: successes, setbacks, paradoxes and perspectives

RBMOnline - Vol 15. No 5. 2007 582-590 Reproductive BioMedicine Online; www.rbmonline.com/Article/3118 on web 27 September 2007

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!BSTRACT The present review gives a subjective outline of the past and future of somatic cell nuclear transfer (SCNT). The first decade was full of contradictions: amazing successes were followed by frustrating fiascos. Although the possibility of reversing somatic cell differentiation completely is a more or less acknowledged fact, the underlying mechanisms are obscure. Consequently, the advancement has been mostly empirical and rather slow. Efficiency is slowly increasing in some species while stagnating in others, and the technology is too expensive for most practical purposes. The number of cloning laboratories is rather small, and those with reasonable productivity are extremely rare. SCNT research is underfinanced because of the atmosphere of suspicion surrounding cloning and the controversial reputation of cloners. However, certain signs may indicate a more successful next decade. In some species, technical refinements have resulted in a considerable decrease in developmental anomalies. Even the actual efficiency seems to be suitable for special purposes with high scientific and commercial impact including transgenic domestic animal production for human disease models, xenotransplantation and biopharming. Human therapeutic cloning is now a realistic perspective, and certain responsible scientists have started to question the reasonableness of an eternal ban for human reproductive cloning. Just one fulfilled goal out of the many promises of SCNT would justify the invested efforts. Keywords: cloning, domestic animal, ethics, human, reprogramming

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The first mammal cloned from adult somatic cells was a sheep born in Scotland on 5 July 1996. Compared with the extraordinary importance of the event it is difficult to understand that the publication was delayed by more than 7 months (Wilmut et al., 1997). In the 16th century, just decades after Gutenberg, scientific discoveries were printed and distributed at the same rate throughout Europe. Various factors including confidentiality before patenting, obtaining evidence of origin and health, writing of the manuscript, and the review and publication process might have played a role in this delay. However, although this lead time is unfortunately rather typical and not at all exceptional in scientific publications, all these

problems should have been tackled in days, maybe weeks, in the case of an unquestionable breakthrough. Accordingly, the 7 months may also indicate that neither editors nor authors were completely aware of the significance of the event and especially the subsequent reaction of scientists and laymen worldwide. The original goal, cloning from a cultured cell line, had already been achieved and published earlier (Campbell et al., 1996), and the new success was probably regarded as a logical continuation. The length of the paper (just four pages in the form of a letter), and the almost hidden announcement of the achievement, with only a few modest words commenting on its value, also support this assumption.

© 2007 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK

Review - Somatic cell nuclear transfer - G Vatja

However, the world in 1997 was hungry for exciting news. We were over communism and still before 9/11, between two Balkan wars, and ad interim, Americans did not want to occupy Iraq. Additionally, the topic – for the media – was more than fortunate. A typical tendency in the second half of the 20th century was the dethronement of ideas, faiths, heroes and hierarchies. Science has suffered enormous casualties, and, to be quite frank, not without real reasons. After the glory of Gagarin and the Apollo mission, we had the Challenger and Columbia: series of seemingly insignificant tragicomic mistakes that eventually discredited the whole space programme. The reputation of human medicine was also badly eroded. The start was probably the Contergan scandal (Botting, 2002), followed by a series of unfulfilled promises and lost individual battles (radical expansion of lifespan, cancer therapy, etc.). The increasing public interest in ‘alternative’ treatments clearly demonstrates this fiasco. Scientists, respected a generation earlier as half-Gods and creators of a bright future, had become parasites and suspects. In this regard embryologists have a privileged position, in a fully negative sense. This branch of science has been disgraced first by Huxley’s ‘Brave New World’, and indirectly damaged subsequently with the eugenic ideas and practices before and during the Second World War, although no definite mammalian embryology existed at that time. Later, Hollywood’s contribution dominated: after Frankenstein and Dracula, cloning has become the favoured subject of horror and sci-fi movies. With almost no exception, clones were (and still are) characterized with fully negative features and have to be killed like worms; their creators are the typical mad scientists with high intellectual capacities but evil intentions. Unfortunately this emotionally charged view is shared by the most distant groups of various societies regardless of age, sex, education, and political or religious affiliations. Considering all these facts the elementary harsh reaction was predictable, although at that time nobody could predict it. Or was the delayed and modest announcement the sign of a (partly subconscious) fear of the subsequent events? Journalists have also accused embryologists with euphemism for the term ‘somatic cell nuclear transfer’. This is a rather malign charge, without any real base. Although, for the layman, cloning is just making identical animals or humans, in biology its meaning may be completely different depending on the discipline: cell culture, molecular or reproductive biology. Even in embryology, cloning can be performed in various ways, for example by embryo splitting, or using donor cells from another embryo. Somatic cell nuclear transfer (SCNT) is just one of these cloning techniques, even if its theoretical and practical significance is far above the other ones. Although SCNT in mammals is only a 10-year-old technique, its roots go back to the 19th century. The first primitive but successful nuclear transfer experiments were performed before the First World War (Driesch, 1892; Loeb, 1894; Spemann, 1914). The final version was theoretically outlined before the Second World War by a German (Spemann, 1938), and realized (without having any idea of the previous outline) by Americans after the war (Briggs and King, 1952).The fact that this principle was only used successfully in the 1980s in mammals (with embryonic cells as donors; Willadsen, 1986) can be attributed to RBMOnline®

the inappropriate methods for handling mammalian oocytes and embryos rather than the technical realization of nuclear transfer. Finally, the decade delay between embryonic and SCNT was definitely the result of a psychological barrier. Retrospectively, there was nothing to hamper the application of the somatic cell nuclear transfer technique, used successfully during these 12 years, to embryonic cells, except for one factor: nobody had the courage and extravagance to try it. This statement does not diminish the merits of the creators, in contrast it stresses the value of their approach and the significance of their achievement. It should also be mentioned that with the recent simplification of SCNT, the work could be done most probably before the First, but definitely before the Second World War. The latest sophisticated instruments of embryo technology including expensive inverted microscopes, highly accurate micromanipulators, laser beams and piezo techniques can be useful for some special purposes, but SCNT can also be performed with a simple stereomicroscope, an inexpensive impulse generator, with a not-too-sharp blade, and a mouth pipette (Vajta, 2007). Foil bags filled with the appropriate gas mixture and submerged in water-baths can perfectly replace computer-controlled incubators (Vajta et al., 1997). Paradoxically, the principle was perfectly outlined long ago, the manual skills of scientists were probably much better than today, the instruments were available, but to put things together, to find the right solution for small details, required a struggle for two generations.

7HATISREALLYGOINGON In spite of the thousands of offspring born in various species during the past decade, the subcellular and molecular biological mechanism of SCNT is almost completely obscure. Embryologists are just amazed it can happen – maybe even more than other scientists and laymen. Professionals in this field know perfectly well what sophisticated mechanisms are used by nature to prepare gametes for fulfilling the ultimate task. Centuries of struggles to make in-vitro analogues, hundreds of millions of infertile couples prove how sensitive this system is; how many factors have to contribute in a perfect orchestration to achieve fertilization, to start embryo development? Some scientists who marvel at the delicate nature of the process, state even today that the only way to establish successful assisted techniques is to learn and re-establish in-vivo events in the laboratory. Unfortunately, this attractive idea provided little if any help for improvements of in-vitro systems in reproductive biology. One typical example is the oocyte:spermatozoon ratio at fertilization. In vivo in the oviduct, it is eventually very close to 1:1 as a result of the repeated barriers established by the joint effect of the female genital track and the demanding capacitation process of sperm cells (Hunter, 2003). In vitro, however, efficient fertilization requires 10,000–100,000 spermatozoa per one or a few oocytes. The need of the supposed in-vivo selection (if there is a real selection at all) can also be questioned according to the success of in-vitro fertilization and especially intracytoplasmic sperm injection (ICSI). Another popular idea is the use of oviductal or cumulus cells as feeder layers to support embryo development in vitro, with specific factors that are produced by these cells in vivo. This



Review - Somatic cell nuclear transfer - G Vatja

approach resulted in some modest improvement 10–15 years ago (Gandolfi, 1987; Conway-Myers, 1998); however, the more recent application of simple medium with some selected essential components provides much better results regarding both qualitative and quantitative measures (Gardner, 1998; Holm et al., 1999). Most probably we also underestimate the tolerance and flexibility of gametes and the process of fertilization. The number of abortions worldwide (approaching 100 million, considering only the legal ones during the past decades) and the success of latest assisted reproductive techniques (including ICSI, preimplantation genetic diagnosis and cryopreservation) prove that fertilization may also occur against our best intentions or with the most drastic violation of the natural processes. Gametes are strange creatures. The resistance of spermatozoa is simply amazing, and can be perfectly illustrated by the fact that their fertilizing ability (with some support) can be preserved even after lyophilization (Wakayama and Yanagimachi, 1998). In contrast, the oocyte, the key player in SCNT, is commonly regarded as one of the most sensitive cells of mammalian body: even a slight fluctuation of temperature may cause irreversible damage – we had to learn this lesson well as a consequence of the frustrating 70 years when embryology achievements in amphibians could not be reproduced in warm-blooded animals. However, oocytes may be surprisingly tolerant to some impacts. Most entrants into embryology are surprised when they see oocytes vortexed, but this strange and drastic mechanical treatment that has absolutely no in-vivo analogue facilitates rather than hampers further development. Astronaut centrifuge trials have proved that 4–12 g (depending on position) is probably the maximum gravity tolerance for a healthy human being, and the absolute record for scuba diving is slightly below 300 m depth, i.e. approximately 30 bar (3 × 103 kPa) pressure (requiring more than 6 h decompression afterwards). On the other hand, porcine, and most probably other mammalian oocytes, can easily survive 12,000 g centrifugation for 20 min (Du et al., 2007a) and 400 bar (4 × 10 kPa) pressure for 60 min without any need for stepwise decompression afterwards (C Pribenszky, personal communication). Even more surprisingly, these effects may rather improve, and do not compromise, their developmental competence. Their protein shell, the zona pellucida, can be punctured, cut and digested. They can be bisected, fused, microinjected, and parthenogenetically activated with various harsh interventions. If these processes are done appropriately, they survive and develop to the stage that the genetic information allows them to.



The most astonishing thing, however, is what happens after SCNT. For practical reasons oocytes are usually derived from ovaries of slaughtered animals and matured in vitro, i.e. supposedly their quality is intrinsically handicapped. Subsequently, they are subjected to many of the above-mentioned procedures (vortexing, slitting of the zona pellucida, removal of nuclei or zona digestion, and manual bisection, depending on the method), and unified with the somatic cell through fusion or microinjection. If they are lucky, at this point a short period of time is offered them; however, this is not for a rest – quiet relaxation is the privilege of the cloner, not the reconstructed embryo. Supposedly, the period between reconstruction and activation (if protocol used allows such period at all) is used

for reprogramming. If the SCNT is done using other protocols, the reconstructed embryos have to be reprogrammed in parallel with other substantial changes. Reprogramming is the key event of SCNT – and the least understood one. The start and the end are clear. A fully differentiated nucleus regains totipotency, i.e. the ability to govern embryonic development from the very beginning. In a few hours, its genome has to replace the joint function of that of the oocyte and spermatozoon. Maybe the astonishing success of SCNT opened scientists’ eyes (again) to how little of the molecular basis of normal fertilization and early embryo development was known. Some great achievements of the last decade, including the decoding of DNA, might serve as a basis for understanding, but the key mechanism is probably the epigenetic regulation that determines which genes are expressed and which are not. According to mouse models, it is supposed that a genome-wide release from the block of function of most genes occurs in two subsequent phases: one during gametogenesis, the other during the first days of embryo development. One of the greatest mysteries of developmental biology is how these processes, especially the second one so precisely timed, can be orchestrated and performed including differences between demethylation in the paternal and maternal genome, and the special handling of imprinted genes. All these tasks should be performed alongside the basic structural changes – cleavage, compaction and early differentiation at blastocyst formation – that occur in this very short period of time. Disassemble the most sophisticated blueprint we know, re-design it for the new situation and rebuild it in several hours or a few days: the achievement matches that of a wandering circus. The intensive worldwide research in particular fields has not helped much in clarifying the overall view, so far. Most investigations have been performed in vitro, the majority of them in mouse, and general conclusions have been made accordingly. However, mechanisms involved may be basically different from species to species, even between close relatives. Genome-wide active demethylation of the male pronucleus happens quickly in mouse and human, at a more moderate rate in pigs, cattle and rats, and seems to be missing in sheep and rabbit (Morgan et al., 2005; Renard, 2005; Zaitseva et al., 2007). Eventually, apart from the correlations, there is no direct evidence proving that global demethylation in preimplantation embryos is indeed essential (Arányi and Páldi, 2006). Methylation patterns are influenced much by the actual in-vitro environment (Wrenzycki and Niemann, 2003). Timing and mechanisms of activation of the new genome also show huge variations between species, as documented by ultrastructural and molecular biological observations (Hyttel et al., 2000; Renard, 2005). Most scientists have regarded, and many scientists still regard, SCNT as an extremely rude intrusion in this delicate and barely understood process; a hopeless attempt to change one of the basic laws of nature. Offspring born were and still are explained as lucky exceptions. The overall opinion – shared also by some cloners – is that we only can expect advancement (if any advancement can be expected at all) with a thorough understanding of the molecular mechanisms. However, during the past 10 years the overall efficiency of SCNT has increased steadily regarding offspring per oocyte and expense RBMOnline®

Review - Somatic cell nuclear transfer - G Vatja

per offspring, while the frequency of abortions, stillbirths and developmental abnormalities has decreased in some species and laboratories to a perfectly acceptable level (Renard, 2005; S Davis, personal communication; C Galli, personal communication; H Niemann, personal communication) even for the most scrupulous bioethical surveillances. Surprisingly, the contribution of basic science has been minimal in this process so far. Most improvements have been achieved with empirical fine-tuning of the original techniques, just like in case of the IVF and other embryo technologies. With every passing day, new and new living evidence proves that SCNT is a biological reality. Based on the rate of advancement one cannot completely exclude a future where the efficiency of SCNT will be comparable or superior to that of IVF or even natural fertilization. How can we solve the sharp conflict between the ‘theoretically impossible’ versus ‘in practice it works quite well’? One explanation may be that the natural reprogramming process is not as delicate and complicated as originally supposed. As mentioned above, most of the available data have been derived from mouse, and when conclusions are made, considerable differences between species are simply forgotten or not emphasized enough. Additionally, the detected mass changes including demethylation–methylation may be regarded as summaries of precisely determined specific processes; however, they can also be explained as a tendency, where individual differences between embryos and cells of embryos may and do exist (Arányi and Páldi, 2006). The relatively few available data regarding specific gene functions in single blastomeres may support the latter possibility (Bodó et al., 2005). Evidently, this tendency may offer much more flexibility than a Swiss-watchlike precision, even for such drastic interventions as SCNT. Another possible explanation is that deprogramming– reprogramming is really as delicate as most scientists suppose, but there is a widespread and efficient repair and correction mechanism that helps to eliminate mistakes. Biology is full of such protective mechanisms. In multicellular organisms the immune system protects the individual on at least at four different levels. The pH of body fluids is maintained with multiple buffer and compensation systems. Also in molecular biology, efficient corrections eliminate the unavoidable mistakes including the repair of DNA strains at several checkpoints during the cell cycle. It is more than likely that similar mechanisms operate at the epigenetic level, where at least some part of the processes discovered and detected appears to be vital for the future development and differentiation. More explanations may exist, and more than one may be correct. The undeniable fact is, however, that there is a fantastic flexibility in reversing the process of differentiation in mammals, at least on the level of the oocytes and preimplantation embryos (Renard, 2005). We have other evidence for this flexibility. Although the fate, i.e. the direction of differentiation of mouse blastomeres, is determined as early as during the first or second cleavage (Edwards, 2005), the possibility of embryo splitting and chimera production by aggregation proves that blastomeres may disregard these early instructions and restart distribution of roles. The success of fertilization with intracytoplasmic injection RBMOnline®

of round spermatids, unprepared and incapable of natural fertilization, also proves the ability of the fertilization–early development process to adopt to new, unexpected situations (Ogura et al., 1994; Tesarik et al., 1995; Yanagimachi, 2005). However, beyond doubt, SCNT is the most extreme and most surprising evidence to this almost limitless flexibility. Almost limitless: but there are still some limits as illustrated by the fiascos and frustrations during the 10-year history of SCNT. By summarizing the available (insufficient and controversial) data and the opinions of experts in this field, the reorganization of the epigenetic regulation of the DNA is done in a similar way as in normal fertilization, but in a compressed manner with variable efficiency (Gao et al., 2007), and with huge individual differences between embryos even from the same batch of cloning (Somers et al., 2006). It should be more or less complete before the first definite differentiation step, the trophoblast–inner cell mass separation during the blastocyst formation (Renard, 2005; Alberio et al., 2006). Afterwards, the inner cell mass may still remain flexible and ready to heal, but the trophectodermal cells may become handicapped, with many possible consequences in the formation of extraembryonic tissues including the placenta. Many (although not all) of the SCNT-related syndromes including abortions, stillbirths, large offspring and late anomalies are supposed to be related to this stage of the process. Another crucial source of problems is the evident confusion with imprinting, and possible shortening of telomeres, although – according to recent investigations – this problem is far less serious than supposed earlier (Schaetzlein et al., 2006). On the other hand, the good news is that – in spite of the frighteningly disorganized morphological appearance that may occur soon after nuclear transfer – chromosomal abnormalities seem to play a small role in developmental problems. Blastomeres with chromosomal deficiencies may be replaced by healthy ones; on the other hand, if the anomaly is widespread, the embryo may die before or soon after implantation. The relative intactness of the genome, as well as the extremely efficient re-setting of the epigenetic landscape at gametogenesis, are also proved by the next generation where no increase in developmental abnormalities compared with control offspring has been observed, even when both parents were produced by SCNT (Tamashiro et al., 2002; Heyman et al., 2003; Wells, 2003). There have been many attempts to facilitate the reprogramming process directly, including the use of oocyte extracts or defined chemicals; however, only a few have been successful and none of them has obtained a widespread acknowledgement yet (Alberio et al., 2005; Sung et al., 2006; Kishigami et al., 2007; Miyamoto et al., 2007). So far, any attempt to replace the originally used metaphase II (MII) oocytes as recipients has resulted in compromised development. For somatic cells, the popular hypothesis is that less-differentiated ones evidently are better donors. However, comparative investigations do not support entirely or definitely disprove this idea (Sung et al., 2006; Inoue et al., 2007). Retrospectively, almost all improvements of the past 10 years in SCNT have been obtained intentionally or spontaneously by a simple strategy: to help the oocytes and embryos to resolve the problem by themselves. The keyword here is ‘help’: not to impose additional stress, i.e. to provide the oocytes and embryos with what they really need



Review - Somatic cell nuclear transfer - G Vatja

(optimal temperature, proper media, and appropriate timing of processes) and minimize the unavoidable shock with rapid and considerate manipulations. Although this strategy seems to be obvious even for laymen, it is partially or entirely disregarded by many cloning groups all over the world. The secret of the few really successful ones lies in these simple details. A perfect example that this approach is far from fully exploited yet is the amazing success story of porcine SCNT. Basically, pigs have never been the preferred subjects of laboratory embryologists due to the extreme fragility and sensitivity of porcine oocytes. The moderate interest is demonstrated well by the fact that in contrast to the hundreds of offspring in cattle and sheep, only a single successful embryonic cell cloning (with blastomeres as donor cells) was reported before the era of SCNT (Prather et al., 1989). However, the possibility of making transgenic pigs with the new cloning method fascinated scientists and especially industry, and considerable sums of money were invested into research. After years of desperate attempts, three groups reported success in 2000 (Betthauser et al., 2000; Onishi et al., 2000; Polejaeva et al., 2000). Although one approach included a significant modification of the basic technology – using a twocycle nuclear transfer – this approach was later abandoned, since the one-cycle traditional way has become highly successful, enabling up to 50% blastocyst per reconstructed embryo rates, 50% pregnancy rates after transfer, and minimal losses during and after the pregnancy (Lagutina et al., 2006; Du et al., 2007b). Factors leading to this success included appropriate timing, rapid and skilled manipulation, the right choice of method and adjustment of parameters for activation, and the use of the appropriate medium for embryo culture. A breakthrough in facilitating the reprogramming may happen any day in the future. However, even without that breakthrough, right now we have ample possibilities to establish and/or improve SCNT by discovering the right way to handle oocytes and embryos in many species where the existing methods are inefficient or completely unproductive so far, i.e. in most mammals, including humans.

&UTUREPOSSIBILITIES



Theoretically, it is hard to overestimate the significance and potential application areas of SCNT. According to some opinions, it is easier to list the fields of biology where SCNT cannot be used for several purposes. However, according to the suspicious or rather hostile atmosphere it is surrounded by, the vast majority of these potential application fields have been unexploited and disregarded. Basic science could use SCNT for the simplest purposes (the almost identical genetics – excluding the mitochondrial genes – would decrease the variations in experimental groups providing much higher accuracy to a given test) to the more sophisticated ones, either using the distinctive features of SCNT (the role of mitochondrial genes, epigenetic regulation, differentiation and dedifferentiation, interspecies nuclear transfer, etc.) or profiting from the possibilities it offers (for example the role of epigenetics in cancer formation and possible reversion of the process; by using transformed cells as somatic cell donors). Strangely, apart from a few attempts in this direction, the progress is negligible. One – not insignificant – reason for this paradox is that many serious scientists still prefer to keep a distance from the obscure company of cloners,

and grant applications containing the word or synonyms of cloning may be handicapped if evaluated by conservative referees. Regarding practical applications, the most commonly mentioned possibility is to use SCNT in domestic animals to rapidly propagate some favourable feature (meat quality, milk production, disease resistance, etc.). With the recent favourable risk assessment by the US Food and Drug Administration (2007), a considerable advancement is foreseeable in this direction, at least in certain areas of the globe that may follow the American tendency, excluding the European Union. Once approved and welcomed also by consumers, and with the foreseeable rapid decrease of production costs, there is a great prospect for this application especially in species where the reproductive cycle is long and the litter size is small, predominantly in cattle. In developed countries, cloning of top-quality bulls for semen production seems to be the financially rewarding goal, while in developing areas a rapid improvement of the average stock could be achieved. Immediately after the first reports of successful cloning, the technology was also used for a generally accepted purpose, for preservation of endangered species or breeds. However, apart from some very limited successes, these possibilities have not been exploited and even cloners doubt now if this area can be included in the immediate goals. In most situations, there are far simpler ways for preservation; however, limited financial resources hamper their application. Moreover, for species preservation, interspecies SCNT would be needed. Although some limited successes have already been achieved between very close relatives, it seems to be more demanding than expected earlier, and the advancement is rather slow. Curiously, the most fascinating practical application field of SCNT is the result of a by-product (although this by-product was the original goal when the process was established): in domestic species, this method is the most reliable and efficient way to produce targeted modifications in the nuclear DNA. In contrast to mouse, where embryonic stem-cell-based chimera technology offers an elegant way to make directed changes in the genome, no such possibility exists in domestic animals, due to the lack of a method to produce embryonic stem cells from these species. Alternative methods have been published repeatedly every year, but none of them seems to be competitive with SCNT so far, in spite of the limited efficiency of the cloning procedure and also the limited available time-frame (determined by the lifespan of a somatic cell culture in vitro) to perform the required genetic modification (Robl et al., 2007). When considering the potential application of transgenic domestic animals, human medical research and medical practice are the primary fields. Living bioreactors producing pharmaceutically important proteins in milk, serum or urine were the first target of research: production of these proteins is an order of magnitude more expensive in other systems, and the decrease in cost may save millions of lives worldwide (Goldman et al., 2004; Keefer, 2004). The second target and the subject of rapidly expanding research is the production of organs for xenotransplantation in humans. While the need for organs increases at 15% per year due to the increasing lifespan, improving surgery techniques, better anti-rejection medication and also because rising living standards in some countries provide access for more and more patients to these interventions, the number of available organs remains stable (Vajta et al., 2007). RBMOnline®

Review - Somatic cell nuclear transfer - G Vatja

To overcome the immunological barriers requires multistep modification of the pig genome, but the advancement in this field has been accelerated considerably during the past years or months. According to some estimates, the first clinical trials may be performed in less than 10, maybe 5 years. The third goal was disregarded for a long time, but is considered today as one of the most important application fields: production of models to study human diseases. Both basic medical research and the pharmaceutical industry need appropriate models to study the pathobiology and potential therapy of diseases. Traditional laboratory animals are inappropriate for the purpose because of the small size, limited lifespan, and different genetics and physiology (Vajta et al., 2007). Coincidence of three main factors facilitates rapid advancement in this direction. Firstly, this area is characterized by both scientists and laymen being less hostile to SCNT. In contrast to food, health is getting to be a more and more central problem to citizens of developed countries. One human life – if it is that of a close relative or our own life – is more important than that of thousands of animals, even if they are produced by SCNT. Secondly, this area is financially well supported by both research grants and commercial ventures, especially pharmaceutical companies. Thirdly, as a lucky coincidence, both for xenotransplantation and disease models, pigs seem to be the most appropriate species. As described previously, a very impressive advancement in porcine SCNT has occurred in the past few years. This progress has been mainly based on moral and financial support, but the latest achievements have generated even more support, further accelerating research and promising early practical application. In spite of these positive advancements, we cannot talk about a rapid increase of the number, and remarkable improvement of the financial situation of cloning laboratories worldwide. However, most of the few that survived the devastating period at the beginning of the new millennium did so because of their biomedical collaborations, including in many cases a sharp turn to porcine SCNT, while disregarding other scientifically attractive goals.

(UMANCLONING Although part of future possibilities, this subject probably deserves a separate section, being the ultimate battlefield between supporters and opponents. The intensity of fighting (in arguments, actions and enactments) is most probably unique in the history of modern science. The dominating factor is the emotion. Opponents of human cloning, i.e. the overwhelming majority of humans, are horrified at the idea per se, and desperately look for rational reasons to explain their prejudice. According to our present views, two main purposes for human SCNT exist: the production of patient-specific embryonic stem cells for therapeutic purposes (therapeutic cloning) and creation of humans (reproductive cloning). Apart from the goals, the applied techniques, as well as the definite and potential consequences, are fundamentally different. Only rabid opponents or fanatical supporters try to merge the two approaches. Most professionals, as well as many open-minded, sensible, rational and educated laymen (including political leaders of several countries), make a clear distinction between RBMOnline®

therapeutic and reproductive cloning, and establish their balanced position accordingly. This balanced position may, however, be basically wrong. Support of therapeutic cloning in parallel with the categorical dismissal of reproductive cloning may turn from a seemingly rational compromise to a trap requiring decades to escape from. Without doubt, therapeutic cloning is the easier issue. Accidents, deadly or devastating diseases, may occur anytime to any of us, our children or our parents, creating a tragic situation without any realistic hope. Stem-cell research is expected to provide an escape route to many of these dead ends. While cloning is the evil, stem-cell research seems to be the saviour in biological research, supported worldwide with orders of magnitude more money, researchers and facilities. Eventually, however, cloning has become an inconvenient but unavoidable partner of this glorious science, as according to the present knowledge, a full reprogramming of the patient’s somatic cells cannot be performed without SCNT. Partial reprogramming may not be enough, and the use of alien stem cells in most situations would not offer substantial benefits compared with the present day organ and tissue transplantation. Cloners, on the other hand, are grateful for the crumbs of glory and money, and they regard the perspective of therapeutic cloning a great reason for their existence. Unfortunately, the only human embryonic stem cell lines from SCNT were not created but faked. One cannot say it was a good start, and the future of the marriage of disciplines may not become better either. In the worst-case scenario one party – or both – will not be able to fulfil their basic duty. There is a real (although slight) chance that SCNT in humans will never be efficient enough to be considered as a source for embryonic stem cells. Research in this field is currently paralysed by the low availability of developmentally competent human oocytes, and the future solution is still unclear. Although oocyte-like cells may be derived from embryonic stem cells or oocyte precursors, and may eventually have the same reprogramming capacity as in-vivo developing oocytes (Hübner et al., 2003; Clark et al., 2004), the advancement in this field is far from convincing. Animal cells as replacements (Chen et al., 2003) may raise a lot of biosafety and ethical problems apart from the questionable technical feasibility. Supposedly patientspecific stem cells may not become entirely patient specific and may induce rejection. Also, there is a real (and not negligible) chance that the whole embryonic stem-cell therapy issue is just a dream; the great tasks including oriented differentiation, control of proliferation and development of complex structures will remain unresolved, and the intervention will become far more risky and far less competent than expected. On the other hand, stem-cell research may find a reason to separate itself from cloning. Patient-specific stem cells may not be needed, allotransplantation of generic stem cells may replace them with new methods to decrease their immunogenicity and/or increase the tolerance of the immune system (Hall et al., 2006). Embryonic stem cells may be replaced in future



Review - Somatic cell nuclear transfer - G Vatja

treatment schemes with tissue-specific stem cells obtained directly from the patient. They may function appropriately or even better for the given purpose. Alternatively, totipotent stem cells equal to embryonic stem cells may be derived from tissue-specific stem cells or differentiated cells without oocytes (Hochedlinger and Jaenisch, 2006; Collas and Gammelsæter, 2007; Fu, 2007). Another recent discovery with limited practical value shows that embryonic stem cell line preparation from single blastomeres is possible; it may offer a solution for a growing fraction of the population where the fertilization is performed in vitro (Klimanskaya et al., 2006). Accordingly, the future of therapeutic cloning is not entirely safe. From the cloners’ point of view, it may not be wise to stake everything on one card, and anathematize reproductive cloning. From the point of view of humankind (and that is of course much more important), reproductive cloning deserves more. In general, none of the natural human activities is surrounded by such a controversial atmosphere as reproduction. This rarely mentioned but easily acceptable fact is probably the reason why infertility is not regarded in the same way as blindness, deafness or other handicaps; rather, it is mentioned tactfully as a ‘state’. The atmosphere also determines the behaviour of infertile couples: most of them prefer to hide the problem, and find private solutions. An estimated 10–15% of fertilityage couples are affected – accordingly infertility is one of the most widespread handicaps or diseases – but their joint activity to defend their interests is surprisingly weak, and the rest of humankind does not overexert him- or herself to help. No groups in a civilized society would declare such nonsense that eyeglasses, hearing aids or wheelchairs are immoral; however, assisted reproduction is again and again a matter of debate. Arguments against human reproductive cloning are related to biological safety, purpose, potential consequences on the child, its close environment and society as a whole. For safety, this is the best and unquestionable argument against reproductive cloning. But when we talk about safety, we talk about safety and not cloning. Today, serious dangers are related to this emerging technology preventing any reasonably thinking professional from transferring SCNT human embryos into the womb of a woman. However, with the actual rate of advancement, human cloning may become technically possible in a foreseeable period – say in one or two generations – with acceptable risk comparable with that of other assisted reproductive techniques, or natural conception (we also have to keep in mind that there is a slight but realistic chance that SCNT will be safer than natural reproduction). We have to start to prepare for that situation right now.



Regarding the purpose, it may of course be evil, but why should it be? With the slippery slope, prognostication media make the extreme reasonable and the reasonable extreme. Anybody anytime may kill with a kitchen-knife; however, it does not happen too often, and the society’s response is not to ban the knife, but to punish those who used it for the wrong purpose. Reproductive cloning would be much easier to control than a knife; it requires a professional team, donors, recipients, special conditions etc. Reproduction including assisted forms is primarily a family business. Just like IVF can be controlled efficiently according to the actual laws (differing profoundly

from country to country worldwide), application of reproductive cloning could also be restricted to certain areas, first of all to help infertile couples (Kunich, 2003; Macintosh, 2005). Even assuming that human reproductive cloning will be safe and used exclusively for infertility treatment, there are a lot of different concerns regarding its potential consequences to dignity, and the possibly emerging psychological and social situation (Levick, 2007). However, according to a recent editorial in Nature (21 February 2007), a consensus is being formed that ‘dignity is not undermined if a human offspring is valued in its own right and not merely as a means to an end’. Other potential consequences to the child, the environment and society are often overestimated, and are parts of a reflex resistance to a new technology (Simpson, 2007). Most of these arguments have already been heard in relation to the test-tube babies 30 years ago. Actually, in Denmark close to 8% of children are born as the result of assisted reproduction; in a foreseeable period every school class will have two such children and the situation is perfectly tolerated by the society. As stated by Strong (2005) each of the anti-cloning arguments ‘involves serious problems that prevent them from being a reasonable objection in the context of the infertility cases considered’. The almost unanimous, unconditional and unlimited refusal of reproductive cloning can also be interpreted as the majority’s attempt to oppress the needs of an unprotected minority. The actual atmosphere does not even allow the members of this minority to realize the possible future solution they are blocked from.

#ONCLUSION SCNT, a technology with enormous future promises and possibilities, has generated unprecedented harsh reaction from all parts of societies. The only comparable past example is in Greek mythology: Prometheus, the Titan, stole the fire from the Gods and taught people how to use it. A cruel punishment was ordered by Zeus. Prometheus was chained to the rocks of Caucasus and an eagle was set to feed upon his liver every day. He suffered because he did something against the wishes of the Gods, against the laws of nature, and provided humans with a very dangerous technology. Yes, fire is a very dangerous technology. It may burn up cities, kill thousands of people. However, during the centuries, we have learned how to use the fire, and we have also had to realize: we need the fire.

!CKNOWLEDGEMENTS The author thanks Adrás Páldi, Zoltán Macháty and Poul Maddox-Hyttel for helpful advice and critical reading of the manuscript.

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Review - Somatic cell nuclear transfer - G Vatja

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