Human cloning

Antonio Pardo.
Published in Dolentium Hominum, nº 36, year 12, 1997, nº 3, pp. 28-31.

Introduction

The recent publication in the journal Nature of article reporting the successful cloning of a sheep from a cell of an adult sheep1 has unleashed a flurry of commentary across the media at speech. The implications of this experiment, both scientific and ethical, are remarkable. However, many of the opinions expressed in the wake of the news lack a good deal of imagination, and require clarification. In order to do so, we will describe the experiment conducted, its background, the scientific conclusions that can be drawn from it, and the ethical implications of its possible systematic application in a future that, until recently, seemed very distant.

Background

The attempt to obtain viable living beings from somatic cells has been in the minds of scientists for quite some time. However, experiments have never produced satisfactory results. At most, tadpoles had been obtained by inserting nuclei from amphibian embryonic cells to replace the original nucleus of the egg or ovum, but no adult specimen had ever developed2.

The usual interpretation of these failures was attributed to the loss of totipotency of embryonic cells very early in the course of development. During embryonic development, parts of the genome are supposed to be activated and repressed, so that the state of the DNA in the nucleus of an adult cell is very different from that of the newly fertilised egg cell; the adult cell is unable to adequately express the full sequence of commands needed for development and morphogenesis.

For this reason, in the experiments that have been carried out, the tendency has been to use embryonic cells, the earlier the better: it is assumed that these cells still have much of the totipotency that is lost in adult cells and are therefore better candidates for successful cloning.

Embryonic fission

The simplest line of work available consists of embryo fission: the division of the embryo into a few cells, so that each of the resulting cells produces a complete adult being. For example, the division of very early mouse embryos has already been successfully achieved in the past decade, producing several embryos from a single embryo.

This line (the employment of cells in the embryonic state) was the one used in the experiment by Hall and Stillman3 in 1993, which also caused a great deal of discussion, mainly because it was carried out with human embryos. This experiment was not particularly technically complicated. The authors took 17 embryos of two to eight cells, left over from the in vitro fertilisation internship : these were not normal embryos, but triploid embryos, result from the fertilisation of one egg by more than one sperm, a relatively frequent phenomenon during the internship of assisted reproduction techniques. These triploid embryos are not viable and were waste material. The researchers removed them from their zona pellucida, micromanipulated them to divide them, and obtained 48 embryos, which they placed in a culture medium containing sodium polyalginate, which replaced the original zona pellucida and allowed further growth of the divided embryos.

The results were as follows: when the original embryo had 8 blastomeres before cleavage, the new embryos developed to the 8-cell stage at most. If it had 4 blastomeres, they could reach 16 cells. And the embryos that resulted from the division at the two-blastomere stage grew to 32 cells, looking good; it is not known whether the latter would have developed further. Hall and Stillman had decided to stop the experiment there. It would have been necessary to implant them in order to continue their development.

Hall and Stillman's experiment had two objectives. The first, theoretical and primary, was to find out whether human embryonic cells at the morula stage really possessed the totipotency usually attributed to them, as was assumed. The experiment, although it seems to have confirmed this assumption, at least for the two-cell embryo stage, is rather debatable in its conclusions: this experiment was carried out with triploid, non-viable embryos; therefore, we do not really know what can happen with normal embryos. We only have the suspicion that the same will happen to them as to triploids, as we already assumed from our veterinary knowledge and from programs of study of spontaneous twinning in humans. In short, the experiment has contributed almost nothing relevant to science knowledge (the possibility of replacing the zona pellucida with polyhalginate gel had already been discovered by Dr. Hall's own team in 1991)4. Moreover, once the first moment of fame, which brought them a award, had passed, serious doubts were raised about the technical and ethical correctness with which the experiments were carried out. In the absence of approval of protocol of the experiment by an independent ethics committee of research , Stillman and Hall had to refund the award received, and were subject to further sanctions.

The second goal of their experiment was practical: to increase the yield of in vitro fertilisation. It has long been known that some women undergoing assisted reproduction techniques do not react adequately to hormonal stimulation, and their ovaries produce a meagre issue of eggs. issue As the efficacy of in vitro fertilisation is linked to the transfer of a sufficient number of embryos, procedure was sought to improve the performance of the technique in those women who react poorly to ovarian hyperstimulation and do not accept donated eggs. This could be achieved by cloning: dividing the single embryo or the few embryos that could have been obtained into several embryos. In this way, these couples with few eggs would have a similar chance of having a child as those who produce many. In addition, cloning the embryos obtained could reduce the dose of hormonal stimulation that women currently receive when undergoing in vitro fertilisation, a stimulation which apparently increases the risk of certain gynaecological cancers and sometimes produces a clinical syndrome that can have serious consequences.

The problem with this technique applied to improve the performance of in vitro fertilisation is its unreliability: given the high issue of dead embryos, even without any manipulation, the cloning attempt can destroy the few hopes of having a child: greed breaks the sack. And it is well known that human embryos are much more delicate than calf embryos, in which the splitting of embryos of selected breeds has been practised successfully (and also with a very poor yield). Embryo cloning does not seem to be a clear solution to this problem.

In addition, cloning was opposed on ethical grounds of subject , largely coinciding with those that have been publicised as a result of the Dolly sheep experiment, and which we will see once the technical aspects of the latter have been described.

The experiment by Wilmut et al.

Although the news that has gone around the world refers to the latest work of research by the team at high school Roslin, the success of their technique was already published last year, although on that occasion the starting cells had been embryonic cells5. The procedure consisted of taking cells and placing them in culture. In successive passages, the nutrient medium was decreased in concentration of nutritional proteins from 10% to 0.5%. In this way, the division of the cells in culture was stopped. Eggs were also taken and their nuclei were extracted by aspiration using a micropipette. As a final step, the cultured cells and the enucleated oocytes were placed on contact and subjected to a brief electric pulse, with two objectives: on the one hand, to create micropores in the membrane of both cells placed on contact, and produce their fusion; on the other hand, to open the calcium channels of the membrane, causing a reaction similar to that caused by the sperm when fertilising the egg, which sets in motion the entire cell metabolism and the development of the new being. This technique was basically the same when embryonic or adult sheep udder cells were used as starting cells, with only the issue number of passages in culture varying.

The yield of the technique was very low: from the fusion of 277 enucleated eggs with the corresponding cultured cell, only 29 embryos were obtained, which were transferred to sheep; from all of them only one lamb, Dolly, was born. As can be seen, this experiment is not cloning as such, since the new living being is not produced from an adult cell alone, but from its fusion with an enucleated egg cell; in any case, the adult specimen obtained is genetically identical to the starting cell.

Scientific impact

The journal Nature itself devotes a article to comment on the scientific implications of result of the experiment6. According to this commentary, its importance lies in the empirical demonstration that tissue differentiation during development does not imply irreversible changes in DNA; the simple "halt" of cellular reproduction seems to reprogramme7 the genetic system, and put it in a position to restart the entire embryonic development until it reaches the adult state.

It is a pity that current prejudices about the role of the genome in development have prevented us from taking the opportunity to go a little further in analysing the theoretical consequences of the experiment. The commonly held hypothesis about the embryonic development assumes that this happens by the programmed activation and repression of various genes involved in tissue morphogenesis and differentiation. The existence of activator and repressor genes has been demonstrated for a few very specific cases. However, embryologists have long known that, contrary to what might be deduced from this purely Genetics hypothesis of development, most tissue differentiation does not require specific substances as inducers. Simple, trivial physical or chemical changes can produce tissue differentiation in the absence of the usual inducer. The action of any drugs or physical agents can interfere with the embryonic development , producing the same malformations, provided they act at the time when the tissue is sensitive to external influence. These phenomena are simply inexplicable through an intricate interplay of activator, repressor, programmer, homeotic, etc. genes, which have, by definition, a specific activity.

By leaning towards the hypothesis of programming Genetics, the current research has closed its eyes to simple phenomena of cellular interaction, of specialization by autonomous progression of cellular functions, associated with homotypic and heterotypic interactions, well known to experimental embryology; it starts to look for in the programming of genes what, with great probability, is not to be found in them. Hence the current bewilderment: geneticists know more and more about genes, but the overall picture of cellular functioning and of embryonic development is becoming more puzzling and obscure by the day8. The current moment of surprise is a privileged time for a critical review of our knowledge about the functioning of the genome during the embryonic development . Let us hope that we do not lack the courage to throw out hypotheses that have been widely accepted until now, but which Dr Wilmut's experiment is beginning to call into question.

Moreover, with a more objective view of the embryonic development , without the current obsession with genetic explanations, some proposals for the application of recent cloning techniques are simply impossible. In particular, it has been proposed that employment of the knowledge that the cloning technique will provide to induce the differentiation of certain tissues from somatic cells. These tissues could be used for grafts and transplants, e.g. skin for burns, bone marrow for leukaemia, nerve tissue for the treatment of Parkinson's disease9. This proposal does not take into account that the only way to induce the emergence of mature tissues from immature ones is their complex interaction with other tissues, as embryologists are well aware: differentiated tissues can only be obtained from a complete embryo. The proposal attempt to discover the keys to programming Genetics and its application to obtain specific tissues is impossible, as it is based on a misunderstanding of the basic concepts of embryology.

Ethical implications

The application of this cloning technique to livestock and its possible application to humans in the relatively near future, after a sufficient period of experimentation, has raised comments, many of them critical. However, these possible applications are not science fiction: Dr Wilmut estimates that significant progress could be made after a couple of years of research10.

In the case of application to animals, the greatest criticism has been directed against the decrease in biodiversity of the cloned species: a herd with unbeatable production qualities of meat, milk, etc. may be obtained. But it would be at the cost of having a very homogeneous population, which could succumb completely to an epidemic, which would affect all the animals in the same way. However, it must also be recognised that such an application is rather problematic from a commercial point of view: it involves the manipulation of embryos and, consequently, less survival of embryos than in vitro fertilisation techniques already used in livestock. The latter are rarely used because of their low success rate, the need to perform them on young cows and only in first pregnancy. Serious difficulties can therefore be foreseen before the technique becomes commercially viable for the improvement of livestock production.

A very different matter is its application to clone very special animals; for example, it has been proposed to clone animals in imminent danger of extinction. More immediately, there is the possibility of cloning animals that have been genetically engineered to produce certain products in their milk that are foreign to it, but useful in human therapeutics. Thus, there are currently sheep and goats that produce factor VIII and other products of therapeutic interest in their milk. As it is quite difficult to obtain a transgenic animal that secretes a certain product in milk, the new cloning technique would avoid having to repeat the manipulation Genetics: it would be enough to clone some of its cells to have an inexhaustible source , without subjecting the animal to an inhumane attention . Along the same lines, we could include the research currently underway to obtain transgenic animals as organ donors for human transplants: although still rather debatable as to its application internship, it is a promising line of research , which could only yield results on a large scale with the incorporation of cloning techniques for the transgenic animals obtained. Another application would be the cloning of animals with a suitable model for a human disease, so that various treatments could be tested in a controlled manner, which is currently almost impossible. Similarly, the issue of experimental animals could be reduced by having exactly the same specimens on which to test the various alternative procedures11.

With regard to human cloning, Dr. Wilmut's own opinion, as that of many other doctors, is firm: although it seems technically possible to carry out human cloning, it should not even be attempted, as it seems an aberration, devoid of clinical utility12. On the other hand, the attempt at human cloning, if it is intended to bring back a deceased person, would only result in a different person, although physically identical to the deceased, like a twin brother or sister born later. This new person would be influenced by their own cultural status , experiences, family, their own life choices, etc. Therefore, it would be pure chance that one would be able to have an Einstein, a great sportsman, artist, etc. again by cloning one of his cells.

From a deontological point of view, one would have to argue, in support of this common sense view, the respect due to the embryonic human being13. If the technique used for cloning results in so many failures (deaths of embryonic human beings), its application is not acceptable until these failures are reduced to a tolerable minimum. On the other hand, as its realisation does not lead to any diagnostic or therapeutic application, its medical application seems unjustified14.

This deontological point of view fits in well with the declarations made in European political circles, which refer to basic human rights as source for the prohibition of human cloning15. In fact, many European countries have banned in their legislation the internship of human cloning (Spain among them), and the European Commission has also expressed its wish to ban the cloning of human beings at European level16.

The problem of its prohibition is more difficult to solve in the US. There, the hierarchy of constitutional values is generally different from those in Europe, with freedom taking precedence over other human rights. Therefore, in order to prohibit a certain activity, whether at state or federal level, it must first be proven in some way that it is harmful to the rest of the citizens, or to some of them. This is the goal of the Commission that President Clinton has set up to study the issue; pending the Commission's decision, the President has banned federal funding to research for the pursuit of human cloning. Incidentally, this ban did not affect anyone, as this research was not being done anywhere.

The problem that arises, in this atmosphere of exaltation of freedom, is that few see the harm inflicted on the child manufactured with it17 . There is no distinction between a child coming into the world and that child being fabricated. In this way, the human right to be born as the fruit of parental love, in a family18 , is blurred, and aberrant manipulations end up being proposed as the most normal thing in the world: in the same way that a family had an extra child to obtain bone marrow for a transplant for their other child with leukaemia19 , it seems coherent that, within this dynamic, already present in the United States, cloning is proposed as procedure to be able to have spare organs, once it is sufficiently effective in achieving its results. For the time being, thank God, the general opinion is almost unanimous in favour of banning human cloning, but only the course of events will tell us whether this wisdom will last.

Notes

(1) Wilmut I, Schieke AE, McWhir J, Kind AJ, Campbell KHS. Viable offspring derived from fetal and adult mammalian cells. Nature 1997; 385: 810-3.

(2) Gurdon JB. Nuclear transplantation in eggs and oocytes. J. Cell. Sci. Suppl. 1986; 4: 287-318.

(3) Hall JL, Engel D, Gindoff PR, Mottla GL, Stillman RJ. Experimental Cloning of Human Polyploid Embryos Using an Artificial Zona Pellucida. Fertility and Sterility 1993; 60 (2 sup): S1.

(4) Kolberg R. Human Embryo Cloning Reported. Science 1993; 262: 652-3.

(5) Campbell KHS, McWhir J, Ritchie WA, Wilmut I. Sheep cloned by nuclear transfer from a cultured cell line. Nature 1996; 380: 64-6.

(6) Stewart C. An udder way of making lambs. Nature 1997; 385: 769-71. The Lancet also comments on these scientific aspects in the same way in the first part of a article publishing house : publishing house. One lamb, much fuss. Lancet 1997; 349: 661.

(7) This is the expression used by Dr Wilmut himself in the summary of his 1996 article , in the body of the 1997 article and in Stewart's commentary to the 1997 article .

(8) Chandebois R. Le gène et la forme ou la démythification de l'ADN. Montpellier: Espaces, 1989; 239.

(9) Winston R. The promise of cloning for human medicine. BMJ 1997; 314: 913-4.

(10) Highfield R. Human clone 'possible in less than two years'. http://www.telegraph.co.uk/. 7-III-97.

(11) Farnsworth E. Multiplicity. http://www1.pbs.org/newshour/bb/science/jan-june97/cloning_2-24.html. 24-II-97.

(12) Roslin Institute. Briefing notes in relation to Nature paper on nuclear transfer. http://www.ri.bbsrc.ac.uk/library/research/nt_notes.html 11-III-1996.

(13) Spanish Medical Association. Code of Medical Ethics and Deontology. article 25.1."It is not deontological to admit the existence of a period in which human life has no value. Consequently, the physician is obliged to respect it from its beginning. ...". article 25.2. "The sick embryo-fetal human being should be treated at agreement with the same ethical guidelines, including the informed consent of the parents, that inspire diagnosis, prevention, therapy and research applied to other patients".

(14) Cf. Code of Medical Ethics and Deontology, article 24.2.

(15) Cf. statements by Noëlle Lenoir, member of the French constitutional committee and president of the ethics committees of the European Commission and Unesco to Le Monde, 4 March 1997, p. 13.

(16) European Commission, Service du Porte-parole. Commission confirms opposition to research on cloning in humans. http://apollo.cordis.lu/cordis-cgi/srchidadb?ACTION=D&SESSION=144401997-3-24&DOC=1. 12-III-97.

(17) See, for example, the pro-cloning opinion of Professor Macklin, who teaches bioethics at the Albert Einstein College of Medicine in Macklin R. Human cloning? Don't just say no. US News & World Report, 10-III-97, 64.

(18) Cf. Sacred Congregation for the Doctrine of the Faith. Instruction The Gift of Life, I, n. 6.

(19) Lehrer J. Multiplicity. http://www1.pbs.org/newshour/bb/science/jan-june97/cloning1_2-24.html. 24-II-97.