Technical and financial hurdles add to ethical and safety concerns over embryonic stems cells while adult stem cells are achieving remarkable clinic successes. Dr. Mae-Wan Ho reports
The first human embryonic stem (hES) cell bank was officially opened in the UK in May 2004 , with Health Minister Lord Warner saying, "This potentially revolutionary research could benefit thousands of patients." The centre contains just two stem cell lines developed by research teams at Kings College London and the Centre for Life in Newcastle. The House of Lords recommended approving human embryonic stem cell research in 2002, the justification for which was to provide cells for replacing tissues in patients with organ failures.
ISIS had already pointed out at the time that research on hES cells was ethically unjustifiable, especially given that adult stem cells, easily obtainable from the patients themselves (see Box 1), appeared just as developmentally flexible as ES cells, and showed much greater promise in the clinic without either the ethical concerns or the risks of cancer from hES cells [2-6].
Research and clinical findings since have borne out all our objections to ES cells, as well as the promises of adult stem cells. There is simply no case for supporting research in hES cells any longer.
What are stem cells?
Stem cells are special cells that can divide indefinitely and give rise to differentiated cells.
There are two main kinds of stem cells: embryonic stem cells isolated from the inner cell mass of an early embryo, which are pluripotent, in that they can develop into all cell types of the embryo; and adult stem cells, found in adults, the best known of which, until recently, are certain cells from the bone marrow that can develop into all types of blood cells.
However, within the past decade, many more stem cells have been found, not just in the bone marrow, but also in the brain, the skin, the muscle, the gut, the liver, and other tissues of the adult; and at least some of these stem cells seem to have as wide a developmental potential as embryonic stem cells.
Bone marrow cells, in particular, were found to give rise to many cells besides those in the blood: in the skin, lung epithelium, kidney epithelium liver parenchyma, pancreas, skeletal muscle, heart muscle, endothelium, nerve cells in the cortex and cerebellum. They have moved rapidly from lab to clinic, especially in repairing damage to the heart after a heart attack (see "Patients own stem cells mend heart", this series).
Another source of easily obtainable stem cells is umbilical cord cells, which have been routinely isolated from the umbilical cord of the newborn for transplant therapy, and has made headlines in successfully treating a woman paralysed for 19 years (see "Cord blood stem cells mend spinal injury", this series).
There are two ways of creating hES cells depending on the source of human embryos, which are destroyed in the process. The first is from surplus fertilized eggs in fertility clinics donated by the parents undergoing in vitro fertilization (IVF) treatments. The second, much more controversial, is embryos created by somatic cell nuclear transplant (SCNT), which gave rise to Dolly the cloned sheep. This involves transferring the nucleus of a cell of an adult (such as the patient requiring transplant) to an unfertilised egg that has had its nucleus removed, which is then stimulated to develop into an embryo. In both cases, the egg is allowed to develop into a hollow ball with inner cell mass, the future embryo, which is harvested and destroyed to create hES cell lines.
The stated advantage of SCNT is that it avoids immune rejection in the transplant patient by using the individuals own genetic material to produce the embryo. It is also euphemistically referred to as therapeutic human cloning, to distinguish it from reproductive human cloning, in which the embryo obtained by SCNT would be allowed to develop further into a live birth, as Dolly was.
Reproductive cloning is now almost universally rejected, mainly because the success rate is extremely low - it took 277 nuclear transfers to enucleated eggs to create a single Dolly and even when successful, cloned animals, Dolly included, invariably suffer many genetic abnormalities and incomplete epigenetic reprogramming (the heritable erasure and re-marking of genes thats crucial to normal development). Currently, the efficiency of nuclear transfer cloning across all species is between 010%, i.e., 010 live births after transfer of 100 cloned embryos . Of about 10 000 genes analysed in mouse clones approximately 400 showed abnormal expression patterns, especially in placentas .
Yet, defective embryos are routinely used to produce ES cells, and positively recommended by some researchers , who stated, "Perhaps genetically deficient cells may be entirely suitable for somatic cell replacement." That is a large assumption, fortunately, not shared by other researchers : "We suggest that the wide range and high incidence of epigenetic defects in nuclear transfer embryos will preclude safe use of this approach [in creating hES cells] until the procedure is dramatically improved."
But if epigenetic reprogramming error is inherent to the somatic nuclear transfer procedure, as pointed out by some researchers , then it is a blind alley as far as tissue replacement is concerned, even if the ethical concerns are set aside. Yet, China, Singapore, UK and USA have already legalized therapeutic human cloning (seeBox 2 ), and Korean scientists reported the first hES cell line created using this procedure in February 2004.
Legal status of ES cells in research
Spare fertilized eggs
Fertilized eggs solely for ES cell research cell lines
Import of ES
The research team in South Korea Seoul National University made headlines in creating the first hES cell line by SCNT . The hES cell line proliferated for more than 70 passages, maintaining normal chromosomes and is genetically identical to the somatic nucleus donor.
Actually, the nuclear donor and recipient were one and the same healthy women who provided both the cumulus cells surrounding the developing oocyte (immature egg cell) and the fresh unfertilised eggs. The nuclei from the cumulus cells were transplanted to the egg of the same individual. A quarter of the SCNT eggs reached the blastocyst stage (at which the inner cell mass is harvested to create hES cells). From a total of 30 blastocysts, 20 inner cell mass were harvested, but only one ES cell line was obtained.
The research, led by Dr. Woo Suk Hwang, was soon mired in controversy . The team had recruited 16 women prepared to have hormone injections to make them super-ovulate, providing the 242 eggs that produced the single hES cell line.
Citizen rights activists and bioethicists complained of the lack of transparency surrounding the recruitment of the egg donors, and raised questions over how rigorously Hwang and his colleagues followed the ethical guidelines laid down for their research. One PhD student, a co-author and another member of the lab were reported to have said they donated eggs, but later denied it, blaming poor English for the misunderstanding. The Korean Bioethics Association has called for an enquiry concerning the recruitment of donors and funding sources .
Even if one sets aside the ethical concerns of using human embryos and eggs as instruments and commodities, evidence has accumulated on the risks and problems of using hES cells that are insurmountable.
There is significant risk of fatal teratoma formations when ES cells are used in transplant , that has been highlighted for many years; and is a major deterrent to progression to clinical trials. This alone has persuaded Germany and Norway to prohibit research on fertilized eggs . The legislation regarding embryonic stem cell research in Norway was recently changed to specifically ban both the derivation and use (including import) of embryonic stem cell lines.
Cross-transfer of animal viruses and other disease agents
All existing lines have been cultured on feeder layers of mouse cells, and are hence unsuitable for transplant, because it risks transferring mouse viruses and other disease agents to human patients and creating an epidemic.
When President Bush gave the green light for research on human embryonic stem cells in 2001, he said federal funds could only be used for research on stem cell lines created before 9 August 2001, and more than 60 were listed. But in fact only 17 are currently available for distribution, and only because the US NIH (National Institutes of Health) Stem Cell Registry was created to document existing cell lines and their availability, and to carry out initial tests to assess of the quality of the lines.
Researchers have created their own hES cell lines since. Douglas Meltons group in Harvard created 17 new lines, but like all existing hES lines, are still grown on mouse feeder cells, so their usefulness in clinical applications will be limited . There have been attempts to develop alternative feeder or feeder-free culture systems, but these were not optimal for deriving and growing clinical grade hES cells, as they all use animal products of one kind or another, and carry the risk of cross-transfer of animal viruses and other disease causing agents .
There are reports of high differentiation rates of hES cells (which destroy their stem cell status) and genomic instability after prolonged culture . For example, some hES cell lines display a certain level of aneuploidy (gain or loss of chromosomes) including the gain of chromosome 17q, chromosome 12 , trisomy 20 (three copies of chromosome 20) or abnormal X chromosome.
There are also frequent epigenetic errors in hES cells. These include differences in the expression of SSEA-4, in telomere length, the down-regulation of collagen, STAT4, a lectin and two genes involved in TGFb signalling, which have been described in different hES cell lines derived in the same laboratory and cultured under feeder-free conditions [18, 20].
Genetic and epigenetic heterogeneity among hES lines
Existing hES cells lines are by no means all characterized. But those that are show considerable heterogeneity even in the same laboratory.
Three different hES cell lines in the same laboratory expressed 52% of genes examined in common, but the expression of 48% of the genes was limited to just one or two of the cell lines . In addition, not all hES cell lines maintain their pluripotency under the same culture conditions, their potential for large-scale culture and growth under feeder-free protocols, or their ability to form teratomas after injection into SCID (severe combined immune deficiency) mice. Moreover, their capacity to differentiate spontaneously into different cell types under in vitro conditions is variable .
The reviewers stated , "To our knowledge there is no study which describes the epigenetic status and stability of different hES cell lines or even one hES cell line after long-term culture." And later, "..it is of concern that application of genetically and epigenetically unstable hES cells in transplantation therapies could be detrimental."
The problem is deeper. Such variability among hES cell lines could mean that knowledge of one cell line would not apply to another line; and worse, if they are unstable in culture, then there can be no possibility for quality control.
No applications in the foreseeable future
Other commentators stated , "Due to the number and severity of the technological challenges remaining to be solved before the initiation of large scale clinical trials, embryonic stem cells are not likely to be a part of routine clinical practice in the foreseeable future."
High costs unjustifiable
The technical difficulties in derivation and culture of hES cells could be expected to involve high costs, especially when these cell lines and procedures can attract patents. It is difficult, therefore, to justify allocation of such large amount of public funds in supporting hES cells research and in maintaining hES cell banks, that could be much better deployed elsewhere; as, for example, in supporting research and development of adult stem cells (including cord blood cells).
Exacerbating health inequalities
Another objection to hES cells research is that it will be a very costly procedure, even if it succeeds, and will exacerbate the global inequalities in access to healthcare . Populations in developing countries have more urgent diseases to fight, and they will be that much more disadvantaged if large portions of the available funds are diverted towards developing hES cell technology by the hype and misinformation surrounding it.
Advantages of adult stem cells
We have stressed the advantages of adult stem cells  as opposed to ES cells two years ago (see Box 3 for an updated longer list). Adult stem cells, in particular, bone marrow cells and cord cells, already have well-established clinical histories; and cannot be patented. They have shown great promise and potential in treating a variety of diseases (see Box 1), including more recently, brain and spinal cord repair in animal models . Adult stem cells can be harvested directly from the patients requiring transplant, and used without culture or after only brief periods of culture, thereby avoid immune rejection and all other technical problems and risks arising from prolonged cell culture. Adult stem cells appear to have all the developmental potential of ES cells - even though the precise mechanisms are debated - without the risks of cancer. On account of the ease of harvesting, handling and use, and the lack of patents, costs are minimal, and hence the treatments developed are likely to be widely available to all. Finally, there is little or no moral objection to using them.
Article first published 16/12/04
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