Embryonic stem cells

and human therapeutic and reproductive cloning

A discussion document for The Royal Society of New Zealand, prepared by Professor R Stewart Gilmour, Chair of Animal Ethics and Biological Safety Committees, University of Auckland, July 2001.

Preamble

The purpose of this document is to provide a basis for further discussions that will ultimately lead to the publication by the Royal Society of New Zealand of a position statement on stem cell research and the wider issues of human cloning. It should be recognized from the outset that within the last five years the national scientific bodies of USA, UK and Australia have already made their positions clear and resolutions have also been passed by a number of multinational organizations including WHO, UNESCO and the European Commission. There is virtual unanimity in the interpretation of the scientific issues which are, in the main, reasonably clear-cut. The ethical safeguards and scientific oversight however are more problematic and worthy of careful reflection. I do not intend to burden this paper with a comprehensive description of embryo development or a reconstruction of the previous submissions. I believe that there is sufficient consensus and clarity of opinion to provide a useful starting point to formulating a balanced view of the potential benefits, risks and alternatives to stem cells in research in New Zealand.

Why stem cells?

Stem cell research is a “cutting edge” area of scientific research. It is anticipated that stem cells will be able to differentiate in the lab into blood, skin and brain cells and may be able to treat cancers, spinal cord injuries, heart disease and potentially many other diseases. It is this unique ability of tissue replacement that has stimulated an intense interest in stem cells as a means to curing degenerative diseases or replacing damaged organs.

Adult stem cells. Certain tissues of the adult human like blood, skin and gut undergo continual turnover and contain a small population of dividing stem cells capable of replenishing the tissue losses as they happen. Other tissues, most notably muscle and liver, have dormant stem cells that only do this in response to tissue loss or damage whilst others like brain appear to lose this regenerative capacity completely. In all cases these cells, known as somatic stem cells, exist in exceedingly small numbers and are able to differentiate into only the tissue to which they belong. Identification and isolation of the individual stem cell types for each tissue of the body is a daunting prospect and it may be many years before they can be harnessed for tissue replacement purposes. Thus, in terms of using adult stem cells for therapeutic purposes the problems are formidable and much research still needs to be done.

Embryonic stem cells. Early embryos, on the other hand, possess a uniform population of pluripotent stem cells that can differentiate into all cell types of fetal and adult tissues. These embryonic stem (ES) cells are isolated from the cluster of 100 or so undifferentiated cells that accumulate in the first few cell divisions of the fertilized egg at which stage the structure is referred to as a blastocyst. In the laboratory, a blastocyst can be split in two prior to implantation to produce two genetically identical embryos and indeed this occurs naturally in humans to give identical twins. This process of cloning by embryo splitting has been used mainly in agriculture to produce clones of sheep and cattle with identical genetic constitutions.

ES cells cannot by themselves form an embryo if transferred to the uterus because they do not have the potential to form a placenta. However if reintroduced into a suitable donor egg and placed in utero they recapitulate blastocyst formation and develop into normal individuals. In contrast to somatic stem cells, ES cells can be manipulated to develop into almost any cell type both in vivo and in vitro. Thus undifferentiated ES cells can be multiplied by tissue culture, cryopreserved until required and then differentiated in culture at a later date or reintroduced into recipient eggs.

ES cells have now been isolated from rodent, primate (including human) and farm livestock species. In the case of the human, ES cells were derived from donated embryos surplus to the requirements of infertility treatment cases.

Cloning by nuclear transfer.

The discussion so far has dealt with the manipulation of cells that have not yet been programmed by developmental influences. An alternative strategy might be to take adult cells and reverse the differentiation process to form the equivalent of pluripotent stem cells. It was thought at one time that when a cell differentiates the characteristics of a particular tissue type (eg. muscle, blood, gut etc.) it also forfeits the ability to reverse this process and thereby return to its undifferentiated state. This notion was proved incorrect almost 40 years ago when it was shown that the nucleus from a gut cell of an adult frog could be transferred to a recipient frog egg (previously deprived of its own nucleus) and eventually develop into a fully mature adult frog genetically identical to the nucleus donor frog. In this process the egg cytoplasm is thought to programme the gut cell nucleus to “dedifferentiate” to form the equivalent of an ES cell that then goes on to divide and form a blastocyst in the usual way. There is no known way of achieving this without transferring the nucleus, that is, so far it has not been possible to duplicate the egg environment in the original adult cell. This was the first demonstration that differentiation is not irreversible and that the genes for early development are not lost or permanently inactivated in adult tissues. It should be noted parenthetically that the failure rate of this procedure is very high probably due to genetic damage to the transplanted nucleus as well as more fundamental and poorly understood problems arising from the gross asynchrony of the cell cycle in donor cells and recipient eggs.

This process, known as cloning by nuclear transfer, has been applied to mammals in more recent times and successful nuclear substitution followed by the birth of live young has been reported for cattle, sheep, pig goats rabbits and mice. Nuclear transfer is therefore the basis of asexual reproduction of cloned offspring that has considerable value for research and animal production purposes. The most publicised example of this is the cloning of the sheep “Dolly” by transfer of a donor nucleus from a mammary gland cell. It is clear from the available details that many of the problems encountered with the original frog experiments still exist today. Dolly was the sole success from 277 attempts. Many of the failures occurred in utero after implantation and the commencement of pregnancy, a finding that was confirmed by similar studies also carried out in New Zealand.

These relatively inefficient and sensational applications however tend to deflect attention away from the enormous potential of nuclear transfer as a research tool to understanding the mechanisms that underlie the differentiation process – experiments that can be carried out in vitro with early embryos and without the need for implantation and progression to term. It is only though this understanding that some day we may be able to regenerate new tissue from existing adult cells without recourse to the embryo and its remarkably versatile ES cells.

Therapeutic v reproductive cloning.

It is clear that the term cloning has now acquired a variety of connotations that depend on the source of the genetic material, its method of propagation and the end point for which the resultant clone is intended. This is an important point because, as will be discussed later, the intent of the cloning has a profound influence on both the ethical justification and public acceptability of the science. In an attempt to emphasise a distinction the terms therapeutic cloning and reproductive cloning have been coined. Reproductive cloning is simply defined as the reproduction of an entire animal from a single cell by asexual reproduction. While cloning by nuclear transfer is legitimately applied to animal production in many countries, it is prohibited worldwide in all existing legislation for the purposes of human cloning. This is justifiable on scientific grounds alone because of safety concerns and the fact that the potential risks are unknown. However, despite the abysmally low success rate of nuclear transfer cloning, the more sinister spectre of its unscrupulous application to eugenics, racist selection and human organ farming represent genuine public fears that need to be recognised.

Therapeutic cloning has been defined as the medical and scientific applications of cloning which do not result in genetically identical fetuses. This definition is somewhat vague as it would seem to include reproductive cloning of a single fetus as an infertility therapy despite the current unpredictability of nuclear transfer cloning. In the UK this issue is addressed by the 1990 Human Fertilisation and Embryology Act (HFE) which requires a license to create an embryo. It also prohibits the development of the embryo beyond 14 days and any attempt to implant it into the womb. It is this definition that is now generally recognized worldwide.

While it may seem reasonable to separate reproductive cloning and therapeutic cloning by definition, clearly they are closely linked operationally and there are no major technical barriers to crossing the boundary from one to the other. Thus there is an important role for legislation to define the present boundaries of acceptability. It is probable however that future scientific progress will remove many of the practical uncertainties of reproductive cloning that make it unacceptable at present. Given that ethics derives in large part from our fear of scientific advances it is important that future legislation evolves in parallel with the new knowledge though reasoned debate at all levels of society.

Stem cells: the power and the promise.

The only realistic treatment at present for organ disfunction resulting from disease or trauma is transplantation, however this is fraught with the problem of shortages of immuno-compatible donors and the need for continual immunosuppression. The prospect that pig organs (xenografts) from immuno-modulated transgenic pigs could be used for human patients now seems less attractive because of the possible transfer of latent porcine viruses and their spread through the human population.

Damaged organs do not always need replacing and repair would be a viable alternative in many cases if a suitable source of healthy replacement cells was available. The aim would be to colonise the host organ with new tissue and restore physiological function without the need for organ replacement. Thus the existing organ becomes a scaffold for tissue reconstitution.

As already mentioned, for adult tissues which contain small numbers of multipotent stem cells, the prospect of adapting these for therapeutic purposes is still distant. Multipotent stem cells have been recognised for only a few of the many tissues of the adult and each is limited to regenerating its own kind. ES cells obtained from blastocysts, on the other hand, can be multiplied in the laboratory before being induced to differentiate into the various types of specialized cells required for tissue colonisation. Two major misgivings have been raised about this proposal. The first relates to the possibility that inappropriately differentiated ES cells may give rise to tumors following colonisation. A solution might be to produce certified banks of stem cells for given tissue types which have been previously screened for chromosomal defects, virus infection and growth abnormalities prior to therapy.

The second problem is rejection. This would not exist if the stem cells were derived from the patient. Current knowledge is not sufficiently advanced to exploit the small numbers of somatic stem cells present in tissue or to de-differentiate an adult cell to form a more primitive precursor. Cloning of a healthy nucleus from the patient by successful transfer to a recipient egg to form a blastocyst however would achieve this end and create a stable line of immuno-compatible ES cells. This would be permissible under the definition of therapeutic cloning in a number of countries however the exceedingly low efficiency of the technique in farm animals suggests that it is not feasible at present in humans. Recent published results from a US commercial animal breeding company describe an improved strategy. Their success rate with reproductive cloning of the cow by nuclear transfer resulted in consistent failure (usually due to embryo death in utero) until they carried out a second transfer of an ES cell nucleus from the newly formed blastocyst to a fresh recipient egg. Approximately 5% of the resulting second round blastocysts successfully developed to term.

While this serial nuclear transfer strategy is not practical from a human therapy point of view, it does suggest that a better knowledge of the reprogramming mechanisms imposed on the nucleus by the recipient egg is a key to future improvement. Basic research is presently being carried out to study these and other controls of tissue determination however realistically it will be some time before we will know whether it is possible to re-programme adult cells so that they revert to being stem cells, or to force specialised cells to replicate themselves. Along the way however much new knowledge will be gained about fundamental biological processes like embryo development and ageing.

The benefits of stem cell research are wide-ranging and can be summarised as follows:

To study the basic processes of developmental and cell biology.
To study the aetiology of some of the most intractable genetic diseases and to develop therapies for them.
To develop revolutionary treatments for degenerative disease by tissue replacement possibly by generating customised stem cells tailor-made to the needs of the patient.
To develop alternative ways of producing pluripotent stem cells without the need to use human eggs including ways to reverse adult cell differentiation and the use of animal (non human) eggs to reprogramme nuclei from adult human cells.
To produce banks of universally compatible stem cells for tissue replacement therapy where the major histocompatability genes and other surface antigens are eliminated and the cells are tested free of pathogenic organisms, chromosomal and proliferative abnormalities.
To aid research and development of new drugs and to provide a source of tissue specific cell types for drug screening, testing and toxicology and to identify new drug targets. The ability to evaluate drug action in human cell lines grown from ES cells would greatly reduce the need for tests in animal models.
To develop improved methods of cloning by nuclear transfer in farm animals to produce high quality genetic stains which are free from diseases including prion diseases associated with transmissible encephalopathies.

A review of international responses to cloning.

Australia.

Research involving human embryos in Australia is regulated by recommendations of the 1996 NHMRC Ethical guidelines on assisted reproductive technology (ART). Specifically it prohibits the following:

Developing embryos for purposes other than for their use in an approved ART treatment programmes.
Culturing an embryo in vitro for more than 14 days.
Experimentation with the intent to produce two or more genetically identical individuals, including development of human ES cell lines with the aim of producing a clone of individuals.

In addition to the NHMRC guidelines, ART is specifically regulated in three States (Victoria, South and Western Australia) where human cloning is an offence. In New South Wales there is draft legislation banning human cloning.

The recommendations of the Council of the Australian Academy of Science in a position statement On Human Cloning (1999) are summarized in the following conclusions:

Council considers that reproductive cloning to produce human fetuses is unethical and unsafe and should be prohibited.
Council is of the opinion that human cells, whether derived from cloning techniques, from ES cells, or from primordial germ cells should not be precluded from use in approved research activities in cellular and developmental biology.

It is instructive to note the moderation of attitudes expressed in these two statements which were made three years apart. To quote from Human Cloning:

For Australia to participate fully in and capture the benefits from recent progress in cloning research, it is necessary to revise the 1996 NHMRC Ethical Guidelines, which, while appropriate when they were written, have since been overtaken by unforeseen advances in biomedical research ……restrictive legislation regarding reproductive technology would need to be repealed in some States……such regulations should be set so as to allow the safety concerns as well as the benefits to be defined.

Council is of the view that there are two potential inhibitors to progress in biotechnology research in Australia. One is unduly restrictive legislation based on misunderstanding of the benefits and the risks; the other is the possibility of a public backlash against science if sensitive cultural issues are ignored by private or public scientific work.

In acknowledgement of a need for more rigorous monitoring to accompany any relaxation of the law, Council made the following recommendation:

It is essential to maintain peer review and public scrutiny of all research involving human embryos and human ES cell lines undertaken in Australia. Council supports the view that a national regulatory two-tier approval system be adopted. Approval to undertake any research involving human embryos and human ES cell lines would need to be obtained from a duly-constituted institutional ethics committeeprior to assessment by a national panel of experts, established by the NHMRC, on the scientific merits, safety issues and ethical acceptability of the work.

United Kingdom.

In the UK, the Human Fertilisation and Embryo Authority (HFEA) has responsibility for the 1990 Human Fertilisation and Embryo (HFE) Act which permits, under licence, research involving human embryos within a period which must not exceed the fourteenth day of development. No licence can be issued for work which is aimed at human reproductive cloning. However the authority will consider licensing research which involves embryo splitting and nuclear replacement for therapeutic purposes. Another body, the Human Genetics Advisory Committee (HGAC) provides broad oversight on the area and reports directly to the British government. In 1998, the HGAC and HFEA jointly issued a consultative paper Cloning issues in reproduction, science and medicine which has led to the setting up by the government Chief Medical Officer of an expert advisory group to look into the details in more depth. Much of their opinion has been garnered from working groups from The Royal Society and a number of useful and informative statements and submissions has appeared. In their statement Wither cloning the Royal Society Council urged that, with respect to research using ES cell lines for the cloning of human tissues, any modification to existing legislation should be carefully drafted so as not to outlaw the potential benefits that could be derived from research on cloned embryos. This multifaceted consultative structure in the UK has considerable merit in that it allows the scientific establishment to be proactive in identifying positive ways in which the legislation can be drafted to avoid exclusion of future research of potential value to medicine. For example, in a later Royal Society statement on Therapeutic cloning the scientific issues relating to successful therapeutic interventions using ES cells are clearly identified as fundamental problems in the fields of cell and developmental biology which may take some years to solve. In the meantime it recommended that a working party be set up to investigate the feasibility of establishing frozen banks of various categories of stem cell that have been both tissue-typed and screened comprehensively for pathogenic viruses.

In a more controversial example, the HGAC/HFEA, after public consultation, advised that consideration be given to the issue of licenses to develop therapies for mitochondrial disease – a condition that could potentially be avoided by reproductive human cloning by nuclear transfer. Although the advice was declined it was incorporated into the terms of reference of a new Expert Advisory Group on Therapeutic Cloning. These examples highlight the importance of productive dialogue between the legislature and the laboratory to ensure that future science best serves the public interest. However most importantly the public must be brought into the debate at an early stage. To this end the Royal Society has published a question and answer paper Stem cell research and therapeutic cloning: an update which clearly outlines to the informed lay person the scientific issues and options which will influence future legislation.

United States of America.

Legislation in the US is bound by the Fourteenth Amendment to the United States Constitution that guarantees the right of procreative autonomy and hence there is no explicit law in State or Federal legislature which prohibits the cloning of human beings. However in 1998, Congress, in making its appropriations for the National Institutes of Health, reinforced an existing law banning all publicly funded research on human embryos with the directive that no federal research funds may be used for the creation of a human embryo for research purposes or for research in which a human embryo is destroyed, discarded knowingly, subjected to risk of injury or death greater than that allowed for research on fetuses in utero. In compliance with this and in respect for the public’s concerns, the federal government did not sponsor research to derive stem cells from embryos. Biotechnology companies however were free to pursue stem cell research with private funds and it was largely due to this effort that the tremendous therapeutic potential for stem cells was realised. Scientists in the public and private sectors are now clamouring to use stem cells to further basic research and to hasten the development of new technologies to treat disease. In 1998 President Clinton received a report from his National Bioethics Advisory Commission (NBAC) on Ethical Issues in Human Stem Cell Research and in response stated:

Because of the enormous medical potential of such research, I have asked the NBAC to look at the ethical and medical issues surrounding human stem cell research. The scientific results that have emerged in just the past few months already strengthen the basis for my hope that one day, stem cells will be used to replace cardiac muscle cells for people with heart disease, nerve cells for hundreds of thousands of Parkinson’s patients, or insulin-producing cells for children who suffer from diabetes.

NBAC recommended that federal funding should be available for research on donated human embryos surplus to fertility treatments as well as primordial germ cells from donated fetal tissue arising from induced abortions. It did not recommend however that federal funds should be used at this time to create human embryos by cloning techniques. Last summer the Clinton administration issued a ruling that the government could pay for research on cells derived from human embryos so long as federally financed scientists did not work on embryos themselves.

In a separate decision, the general council of the Department of Health and Human Services (which controls federal research funding) stated:

The statutory prohibition on the use of funds appropriated to HHS for human embryo research would not apply to research utilizing human pluripotent stem cells because such cells are not a human embryo within the statutory definition.

The final decision, from which definitive legislation will emerge, is now in the hands of President Bush who has prolonged the debate between patient’s advocates and religious conservatives. A number of possible alternative compromises are presently mooted ranging from unrestricted research, banning the further development of new stem cell lines or increased financing for the less controversial but more problematic development of adult stem cells. In the meantime ES cell research continues unhindered in the private sector.

New Zealand.

New Zealand's proposed legislation is embodied in two Bills entitled Human Assisted Reproductive Technology Bill 1996 and the Assisted Human Reproduction Act Bill 1998 which are specifically directed at the control of ART and its associated activities and the establishment of appropriate oversight, licensing and recording of information relating to ART. The guiding principles of these Bills relate to the welfare of any child born as a consequence of ART, the right of informed consent to any of the legal procedures applied, and the rights of individual autonomy and disclosure of one's genetic origins.
Both Bills expressly prohibit the cloning of a human being by any of the techniques described in this paper including reproductive cloning by nuclear transfer. The 1996 Bill also prohibits the creation of a human embryo and the storage or use of a human embryo without a licence from the Human Assisted Reproductive Technology Authority, a regulatory body created and empowered by the Bill. Here the existence of an embryo is taken to begin after the first division of a fertilised egg to form a two cell zygote. In principle this would prohibit the creation and exploitation of human blastocysts for ES cell research and therapeutic cloning without the prior approval of the Authority. Since these Bills are directed primarily at ART, the legal position of ES cell research for therapeutic purposes can only be extrapolated by inference, although the end result is similar to the UK legislation. There is, to my knowledge, no human ES cell research taking place in New Zealand at present however given the enormous biomedical and commercial potential that it offers it is highly probable that ES cell therapy will find an important niche in our national science strategy. It would be useful at this stage therefore to have explicit reference to human stem cell research in the legislation.

Responses from International Agencies and Bodies.

UNESCO. The United Nations Educational, Scientific and Cultural Organisation (UNESCO) states in its Universal Declaration on the Human Genome and Human Rights that “ reproductive cloning of human beings shall not be permitted”.

WHO. The World Health Organisation (WHO) in 1997 adopted a resolution affirming that “the use of cloning for the replication of human individuals is ethically unacceptable and contrary to human integrity and morality”

The European Parliament in its Resolution on Cloning 1997 asserts that “ the cloning of human beings, whether experimentally, in the context of fertility treatment, preimplantation diagnosis, tissue transplantation or for any other purpose whatsoever, cannot under any circumstances be justified or tolerated by any society, because it is a serious violation of fundamental human rights and is contrary to the principle of equality of human beings as it permits eugenic and racist selection of the human race, it offends against human dignity and it requires experimentation on humans… each individual has a right to his or her own genetic identity and human cloning is, and must continue to be, prohibited”.

Concluding remarks.

Legislation as a means of confining the ambit of scientific research is an imperfect instrument of control. Changes in science are often rapid while changes in public opinion (which legislation is supposed to reflect) are less rapid by comparison therefore the balance between promoting or stifling useful scientific research within a legislative framework is delicate. However legislation does not need to be inflexible and should respond to the assurances of new knowledge provided there is appropriate expert review and public debate. In practice however legislation is hard to change and often political expediency preserves the status quo. The experiences of the two leading countries in stem cell research, the UK and USA, outlined above illustrate two quite different approaches to the problem and end up with essentially the same conclusions. A common feature of both is the continual cycle of evaluation and debate (both learned and lay) which contribute to the evolution of public perception and eventually the legislation itself. However a greater challenge lies ahead. In a press release dated 20 June 2001, The Royal Society state that changes to the legislation on therapeutic cloning in the UK may lead to a significant increase in the likelihood that human reproductive cloning will be successfully carried out in other countries where it is not outlawed. They strongly urge that “an international moratorium on human reproductive cloning should be considered by policy-makers because it is the only way to reduce the chances of such experiments being carried out in other countries. But a moratorium must include provisions to ensure that research on stem cells and therapeutic cloning is not jeopardized”.