1° What is an embryonic stem cell?
2° How are embryonic stem cells obtained?
3° What are the applications for embryonic stem cells?
4° What are the problems linked to the use of embryonic stem cells?
5° Do adult stem cells exist
6° What is therapeutic cloning?
7° What are the European regulations regarding stem cells?

 

1° What is an embryonic stem cell?

The human body is made up of several billion cells. Every one of these cells is derived from a single 'egg cell' produced when an egg is fertilised by a sperm. But this does not mean that all our cells are identical. On the contrary, we possess approximately 200 types of cells, with different features and different functions: neurons (cells of the central and peripheral nervous systems), liver cells, skin cells... These specialised cells are said to be differentiated.

Once fertilised, the egg undergoes a series of divisions, yielding two, then four, then eight identical cells. These cells are totipotent, meaning that each one, if isolated and allowed to develop, can form a new embryo. This is how identical twins result from a single fertilised egg.

After the eight-cell stage (two or three days after fertilisation), the cells continue to divide, but lose the ability to form a new embryo. The embryo takes the form of a hollow sphere, known as a blastocyst, containing an inner cell mass. The outer cells of the sphere, together with maternal cells, will form the placenta. The inner cell mass will produce all the tissues of the child. These are the embryonic stem cells (see the diagram) (ESCs). They have two remarkable properties:

• they are immortal: if isolated and placed in a culture medium, they can divide indefinitely, unlike adult cells which stop dividing after a certain time. All the descendants of an ESC constitute an embryonic stem cell line;
  
  
• they are pluripotent: in culture under the influence of appropriate biological and biochemical factors, they can differentiate into any specialised cell type. They can reconstitute the cells of any organ except the placenta: brain, liver, skin, etc.

View the various types of stem cells (see the diagram).

2° How are embryonic stem cells obtained?

In 1998, James Thomson's laboratory at the University of Wisconsin in Madison (US) (http://www.wicell.org/) was the first to obtain embryonic stem cell lines from human embryos at the blastocyst stage, which were produced by in vitro fertilisation (IVF).

IVF enables some infertile couples to have children. At each IVF, multiple embryos are collected, a few of which are reintroduced into the woman's uterus, the others being frozen and stored in case of initial failure. They are stored for several years, even if the couple no longer intends to use them - they may have succeeded in having a child, decided not to have a child, or separated.

These supernumerary embryos (also called spare embryos) obtained by IVF are what James Thomson and his team used to produce the first human ESC lines. Since then, at least three other laboratories (one in the US, one in Australia, and one in Israel) have done the same.

In 1998, John Gearhart's laboratory at the John Hopkins Hospital in Baltimore (US) used a different method to obtain another line of human stem cells. The line was obtained, with the parents' consent, from a foetus aborted at five to nine weeks for therapeutic reasons. The line was isolated from a cell population destined to produce the gonads (testes or ovaries). These so-called primitive germinal cells (PGCs - see the diagram showing the production of PGCs) seem to have the same properties as ESCs.

3° What are the applications for embryonic stem cells?

Biologists value ESCs as an important tool for basic research aimed at elucidating the mechanisms of development and cell differentiation. However, there is another reason for the current interest in ESCs: the prospect of using them for regenerative medicine, i.e., exploiting their immortality and pluripotence to reconstitute organs damaged by disease or accident.

Many human ailments are due to cell degeneration in tissues that doctors are unable to repair. In some cases an organ transplant may be the solution, but in most EU countries there is a shortage of donors. Another solution could be to repair damaged organs with ESCs. The use of cells as therapeutic tools is called cell therapy, as opposed to gene therapy, which uses genes.

Regenerative medicine is still in its infancy. Researchers believe it may take many years to find the right 'chemical cocktail' capable of inducing an ESC able to differentiate into the cell type required. In the medium term, the main applications envisaged for ESC-based regenerative medicine are:

• First, a group of diseases for which transplantation is not an option: the so-called neurodegenerative diseases caused by the death of neurons in precise regions of the brain: Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis... Several European laboratories have already developed foetal neuron grafts - which are not stem cells as they have already differentiated - for the treatment of Parkinson's disease. For more on this, see the following websites: http://ec.europa.eu/research/rtdinfo/en/28/dossier2.html
  http://www.nesu.mphy.lu.se/nectar/NECTARindex.html
  
  
• Some heart conditions might be treated with grafts of ESC differentiated into cardiac muscle cells. The grafts would serve to reconstitute the heart muscle and restore good blood pumping action.
  
  
• Another focus is type-1 diabetes, which represents approximately 10% of all diabetes cases. This disease affects young people and leads to the progressive destruction of the pancreas. It is now possible to slow its progression by transplanting islets of Langerhans, cells specialised in the synthesis of pancreatic insulin, but there are far too few cell donors to meet the demand. Here, too, stem cells might solve the problem of organ donor shortage.
  
  
• By implanting stem cells differentiated into liver cells, it might be possible to repopulate a liver destroyed by a hepatitis virus or by agents that have caused cirrhosis.
  
  
• In the longer term, it might even be possible to reconstruct whole organs of certain types in vitro, and then transplant them into patients. As yet, only the skin, cornea, bladder, blood vessels and bones have been considered for such tissue engineering.

4° What are the problems linked to the use of embryonic stem cells?

In the present state of scientific knowledge, ESCs appear promising for therapeutic applications because they readily divide in culture. Yet care must be taken to ensure that their capacity to divide does not become excessive and lead to tumour formation. Indeed, non-differentiated ESCs transplanted at a certain density can lead to the development of teratomas (embryonic tumours). There is a double risk of tumour development: ESCs are themselves tumorigenic when they are not differentiated; and in mice, the culture cell lines may cause problems of DNA methylation (http://www3.sympatico.ca/diane.demers/methadn/page2.htm). Methylation is the attachment of a chemical structure called a methyl moiety (-CH3), in place of a hydrogen atom, onto one of the DNA bases. This leads to a change in the structure of the DNA and its interactions with proteins, and abnormalities of the karyotype (the chromosomes of the cell). These changes may themselves increase risk of subsequent malignant transformation, that is, the development of a cancer.

Although ESCs are genuinely pluripotent, it is not currently possible to direct them all to form a single type of cell, for example skin cells. As a result, before ESCs can be used for therapeutic purposes, all the tumorigenic cells must be eliminated, and the different cells sorted so that only cells that have differentiated as required for the therapeutic effect are retained. Further study is also needed to avoid rejection of the transplant of ESC-derived differentiated cells by the patient's immune system.

There are currently tens of thousands of stored embryos (the exact figure is unknown) in European IVF centres. They constitute a major reservoir of biological material for producing ESC lines. Yet some scientists are worried about depletion of this supply. They believe that progress in IVF technology will increase the success rate of embryo implantation, which currently does not exceed 30%. Eventually, they say, it may no longer be necessary to produce several embryos in order to implant just one. These scientists believe that in the medium term, spare embryos will become rare and cease to be available for producing stem cell lines. A solution might be to generate spare embryos independently of any parental plan, i.e., solely for research purposes.

The use of spare embryos as a source of 'raw material' (in this case, stem cells) also raises an ethical issue. Some people condemn this reification of the human embryo (treating it like a 'thing'). Others, on the contrary, feel that the very fact that these embryos exist and are stored independently of any parental plan, and the high hopes they raise for treating certain diseases, makes their use ethically acceptable. This is the position adopted notably by the Comité Consultatif National d'Ethique pour les Sciences de la Vie et la Santé (National Consultative Ethics Committee for Health and Life Sciences) in its opinions of 1997 and 2001 (see http://www.ccne-ethique.org/english/start.htm).

PGCs (primitive germinal cells, see question n° 2) seem to divide less readily than ESCs, although the available scientific data are scant. These cells also present an unresolved scientific problem: are they genetically viable? They are taken from foetuses that have been obtained from therapeutic abortions. Some scientists point out that the reason for the abortion may have been a serious malformation or disease caused by a genetic defect, which cell lines isolated from the aborted foetus would be likely to carry. The PGC approach is also criticised by opponents of abortion.

5° Do adult stem cells exist?

As the embryo develops, the differentiation potential of its cells gradually diminishes. In an adult, some cells can produce only one cell type. Others can yield all the cell types of a given organ. The latter are called adult stem cells (ASCs). They are immortal like ESCs, but they are not pluripotent, since their scope for differentiation is limited to a few cell types. The haematopoietic stem cells of the bone marrow, for instance, can only yield blood cells (red cells, various kinds of white cells, and platelets). Such stem cells are described as multipotent. ASCs are also present in certain regions of the brain, in the skin, in fatty tissue, etc.

However, one of the most surprising discoveries of recent years is that under experimental conditions ASCs can also produce cell types other than those they produce in vivo. In 2000, the team headed by Jonas Frisen at the Karolinska Institute in Stockholm (Sweden) (http://info.ki.se/index_en.html ) showed that neuronal stem cells from the brain of an adult mouse can differentiate into kidney, liver, and intestinal cells (this is called transdifferentiation). Other scientists have also shown that haematopoietic stem cells have the potential to regenerate liver or muscle, but with low efficiency. Other bone marrow cells may be able to regenerate the heart, and skin stem cells could be transformed into neurons.

The ultimate goal of ASC-based regenerative medicine would be to take stem cells from a patient's bone marrow, let them proliferate and differentiate into a desired cell type in vitro, and then reintroduce them into the same patient to reconstitute an organ damaged by disease or accident. With such an autograft, there would be no risk of rejection by the immune system as the patient would be receiving a transplant of his own cells.

In addition, the use of ASCs does not raise any of the ethical issues linked to the status of the human embryo. This is why opponents of abortion and of the therapeutic use of ESCs recommend this line of research.

Nevertheless, there are technical obstacles to the use of ASCs. The most problematic is that the ASCs that have been described are only present in small numbers. They may also be hard or even impossible to culture in vitro, as is the case with haematopoietic stems cells. In contrast, skin stem cells, recently shown to have the capacity to be transformed into nerve cells, have the advantage of being easy to culture for long periods.

6° What is therapeutic cloning?

Some scientists believe that one of the most promising approaches in regenerative medicine is somatic cell nuclear transfer (SCNT). This technique involves forming a new embryo by injecting the nucleus of an adult cell into an unfertilised egg deprived of its nucleus. The cell will then develop like a fertilised egg, and the resulting embryo can be used to produce stem cell lines.

SCNT is thus a cloning technique, since it enables a new embryo to be created from the genetic material of a single adult cell. Yet unlike reproductive cloning which aims to give birth to a new being - a practice formally prohibited in all EU countries - SCNT aims simply to produce stem cells for cell therapy. This is why SCNT is sometimes called "therapeutic cloning" (see the diagram).

SCNT thus combines in a single method the benefits of ESCs (easy division and wide scope for differentiation) with those of ASCs (immunological compatibility between the patient's immune system and the grafted cells).

These advantages explain why the UK Parliament voted in November 2000 to authorise somatic cell nuclear transfer for stem cell production. This decision, unique in Europe, followed a report to the Minister of Health (http://www.doh.gov.uk/cegc/) stating that SCNT has "great potential for relieving suffering and treating diseases". The report also recommended that the transfer of embryos produced by SCNT to a uterus should be considered a crime.

The European Group on Ethics (EGE) stated in its opinion of 15 November 2000 (http://ec.europa.eu/european_group_ethics/gee1_en.htm) that "at present, the creation of embryos by somatic cell nuclear transfer for research on stem cell therapy would be premature, since there is a wide field of research to be carried out with alternative sources of human stem cells". This position is justified by the fact that, although there is a difference of purpose between reproductive cloning and therapeutic cloning, there is no difference in nature. There is thus a risk that SCNT might open the way to reproductive cloning.

Another argument is that SCNT requires a woman to donate an unfertilised oocyte (egg), a major intervention requiring hormonal stimulation and general anaesthesia. The EGE stresses the necessity "to ensure that the demand for spare embryos and oocyte donation does not increase the burden on women".

Lastly, some scientists fear that SCNT might lead to the formation of stem cell lines with altered genes. Such genetic alterations could be undetectable in the adult, but produce harmful effects, especially carcinogenesis, after the many divisions that stem cells are likely to undergo.

7° What are the European regulations regarding stem cells?

Europe's ethical and legal pluralism means that it is up to each Member State to legislate on the status of the human embryo and on the use of stem cells.

There is no legislation on embryo research in Italy or Greece.

Research on the human embryo is banned in Germany, Austria, and Ireland. These countries also prohibit the production of spare embryos. In France and Belgium, research on the embryo remains prohibited, but draft bills authorising research aimed at producing ESCs are being studied.

In Spain, Sweden, Denmark, and the UK, research on human embryos less than 14 days old is authorised. Only Denmark and the UK allow the creation of embryos for research purposes. In the latter country, since 1990 this research has been limited to work aimed at improving the efficiency of IVF. Following a report by the Nuffield Council on Bioethics at the end of 2000, the British Government extended this authorisation to include cases where "this research offers substantial hope for the treatment of serious human diseases". Research on ESCs does offer such hope.