Introduction
The plenary policy forum held during the American Society of Hematology (ASH) 44th Annual Meeting, chaired by ASH's President, Dr. Robert Handin[1] of Brigham and Women's Hospital, Boston, Massachusetts, addressed the "state of the science and the policy of the state" on stem cell (SC) research, currently one of the most debated issues in biomedical research and legislative settings. In his introduction, Dr. Handin confirmed that, indeed, this is the most intensely debated and most complex issue that has been discussed at the ASH policy forum since its inception 5 years ago. In his view, political issues are clouding the horizon, and individual states in the United States are now joining the fray in support of or opposition to SC research. These developments are considered "disturbing" in a country that has always been considered at the forefront of biomedical research, ready to develop and advance scientific findings and new medical therapies.
What is the state of the art in SC biology from a clinical perspective? How far have we come in the past few years, and what lies ahead in terms of potential medical advances? Dr. Rudolf Jaenisch,[1] of the Whitehead Institute, Cambridge, Massachusetts, one of the world experts on this topic, gave a very interesting presentation that may provide source for thought for many, scientists, physicians, legislators, and the general public alike.
Cloning of Dolly, the now-famous sheep, is considered in a way a cornerstone in SC biology, but this report was still fresh in print when everybody started wondering what would come next. In the early experiments that led to generation of Dolly, researchers had been investigating whether cellular differentiation involves loss of nuclear potency, and whether nuclei from differentiated cells can be reprogrammed to potency. Reprogramming consists of reactivation of silent embryonic genes and concomitant inactivation of expressed adult genes. Egg and sperm cells are the cells competent to reactivate embryonic genes.[2,3]
The outcome of the fusion process of a somatic (adult) donor nucleus with a somatic SC may yield 3 different outcomes: failure, an abnormal product, or a normal product. By far, the first 2 events are the most frequent outcomes, as this process is marred by high-level inefficiency.
So, the main questions still to be addressed in evaluating the potential use of SC cloning are the following:
What is the potency of donor nuclei?
What is reprogramming?
How can we identify appropriate donor nuclei?
What are the therapeutic implications of SC biology and cloning?
Cloning is performed by isolation and enucleation (by spindle removal) of an egg cell and transfer of a diploid nucleus into such a modified host egg, with formation of a clone that undergoes cleavage of the cloned embryo and implantation in a foster mother. Cloning is a very inefficient process that leads to frequent death of the cloned embryo soon after implantation. The highest survival has been obtained with embryonic SC (ESC) clones, which yield an approximately 15% to 25% survival rate vs 1% with embryos generated from fibroblast nuclei. In addition, owing to their pluripotent differentiation potential, ESCs are easier to reprogram than adult (somatic) SCs (ASCs).
Cloned mice deriving from embryos that survived after implantation showed a very high rate of defects, with fetal overgrowth, placental defects, respiratory distress, and genetic abnormalities.[4] So the question arises, How normal are adult clones, and does the tissue type of the donor nucleus affect quality of the generated clone? Studies performed with both ESC and cumulus cell donor nuclei showed an extreme level of gene dysregulation in output clones and in the offspring, with a faulty expression of at least 4% to 5% of the genome. Of note, 30% to 40% of these dysregulated genes occurred in imprinted genes. The occurrence of such gene dysregulation was related to the nature and quality of the donor nucleus cell, the nuclear transfer procedures, and preexisting epigenetic abnormalities. Consistently, the phenotype of mice cloned from ESCs, Sertoli cells, or cumulus cells showed a high heterogeneity with many de novo abnormalities as well as transmission of parental defects.
Early problems in the offspring are due to dysregulation of key embryonic genes, whereas late complications or abnormalities are due to dysregulation of key differentiation genes. By genetic analysis, 11 genes are active in ESCs and in the embryo but not in cumulus cells. Blastocysts express 100% of these 11 genes when they are derived from ESCs, but only 62% of them when derived from cumulus cells. Thus, ESCs express all embryonic genes and do not require reprogramming, whereas cumulus cells express differentiation genes, but not all embryonic genes and their clones die prematurely.
As demonstrated by experiments using lymph node SCs as nuclear donors, cloning is very inefficient, since clones were generated from a very small number of somatic cells. Of 1040 clones generated in vitro, only 4% yielded blastocysts. Further culture of these blastocysts in Petri dishes yielded expansion of 0.3% ESC lines. Such an extremely inefficient process seemed to work only with the addition of the intermediate culture step. Analysis of the B-cell immunoglobulin (Ig) and T-cell receptor (TcR) rearrangements in the ESCs derived from these cloned blastocysts showed that all mice had clonally rearranged Ig and TcR gene configurations with monoallelic expression and no rearreangements of other Ig and TCR genes.
What are the clinical implications of nuclear cloning? Therapeutic cloning would proceed through 4 steps: cloning of a somatic nucleus, correction of a genetic defect in vitro by gene therapy, expansion of ESCs with the corrected defect, and reimplantation in patients. An experimental model used to evaluate this scenario used the RAG-/- mice, a T-/B-cell combined immunodeficiency model that lacks an enzyme necessary for rearrangement of Ig and TCR genes. ESCs were generated from these mice, differentiated to hematopoietic SCs in vitro and then reinfused in vivo.[5] Low levels of IgG and IgM were seen in the mice engrafted with such in vitro differentiated and "repaired" ESCs.
As mentioned before, 2 different cells types can, however, be used for generation of in vitro SCs, somatic ASCs, or ESCs. ASCs are very rare and very difficult to grow and to differentiate appropriately. It is also not clear whether they would be suitable for gene therapy manipulations, but there are no ethical concerns regarding their use. On the other hand, ESCs occur in very high numbers, are easy to grow and differentiate, and can be easily used in gene therapy approaches, but ethical concernsabout their use have been raised.
Therapeutic cloning with in vitro repair of genetic defects is a very young field, and so far we have no evidence that this strategy will indeed work, but as pointed out by Dr. Jaenisch, "we cannot do away with further research in ESCs, as wished by some politicians."
The availability of nuclear cloning has raised interest in reproductive cloning, which implies generation of a whole new individual by applying this technology in vivo. The very first question that needs to be asked is: Can reproductive cloning ever be safe? The answer is no. Unlike in vitro fertilization (IVF), reproductive cloning would have to reproduce the sequential reprogramming events of gametogenesis. In addition, since the 2 parental genomes are epigenetically regulated, the 2 genomes would have to be separated. How could we ensure persistence of epigenetic differences? Imprinting is not relevant for therapeutic cloning, but it is very important in reproductive cloning.
Advocates of reproductive cloning of humans argue that the procedure could help childless couples to have children, allow very sick or dying patients to be "resurrected," or provide a completely matched tissue or organ donor ("repair kit") in certain instances. However, lessons learned from animal-cloning experiments have shown that this approach is frequently associated with abnormalities and widespread epigenetic dysregulation. In other words, the offspring clone is not identical to its nuclear donor, which defeats the initial purpose.
Commenting on the latest rumors associated with announcements of human-cloning experiments, Dr. Jaenisch deplored such developments, mentioning that they are very damaging for the field. To answer the contention by activists that even IVF, in the beginning, had been strongly antagonized, Dr. Jaenisch stressed that the difficulties associated with IVF were/are only technical, whereas with reproductive cloning, the problems are technical and biological. People should not be misled by the statement that cloned embryos could be screened for normality before implantation. What is "normal"? Can anyone define specific molecular, cellular, and/or functional markers for "normality"? In addition, IVF creates a new life, with a high potential for the embryo to generate a normal baby. Reproductive cloning, on the other hand, is aiming only at propagation of existing life without generation of new genetic combinations, with a low potential for the cloned embryo to generate normal life. Another potential drawback associated with reproductive cloning is that offspring might be generated from the fusion of fertile eggs, therefore giving rise to chimeric individuals.
There is disagreement as to whether embryos acquire human status (become a "persona") immediately after fertilization or immediately after birth. Some believe that only newborns have moral and legal rights. The majority in Great Britain takes the middle position: embryos acquire human status when they undergo implantation in vivo. Thus, the critical issue is not length of in vitro culture of embryos (14 days vs 4-5 weeks). Fertilized eggs have genetic individuality but not personality, as the potential is still there for the generation of twins.
According to the latest pronouncement of the British Parliament, reproductive cloning is a criminal offense, whereas therapeutic cloning is allowed by the law. The critical line that separates them is implantation of the embryo -- in Dr. Jaenisch's view, a very fair conclusion.
Medscape General Medicine. 2003;5(1) © 2003 Medscape
Cite this: Stem Cells: A Plenary Policy Forum - Medscape - Jan 23, 2003.
