During cloning, cytoplasmic factors in the egg cell reprogram the chromosomal DNA of the somatic cell. In rare cases, this reprogramming is sufficient to enable embryonic development to proceed all the way to the birth of a live animal (for examples, see Table 1). In many cloning experiments, epigenetic reprogramming probably fails or is abnormal, and the developing animal dies. Incomplete epigenetic reprogramming could also explain why some live-born cloned animals suffer from subtle defects that sometimes do not appear for years. 12
The completeness of epigenetic reprogramming is crucial for successful cloning-to-produce-children. It will also be important to assess the impact of variation in epigenetic reprogramming on the biological properties of cloned stem cell preparations. If the extent of epigenetic reprogramming varies from one cloning event to the next, the protein expression pattern and thus the biological properties of cloned stem cell preparations may also vary. Thus, it may be necessary to produce and test multiple cloned stem cell preparations before preparations that are informative about human disease or useful in cellular transplantation therapies can be identified.
At this writing, it is uncertain whether anyone has attempted cloning-to-produce-children. Although claims of such attempts have been reported in the press,13,14 no credible evidence of any such experiments has been reported as of June 2002. Thus, it is not yet known whether a transferred cloned human embryo can progress all the way to live birth. However, the steps in such an experiment would probably be similar to those described for animal cloning [see above and references to Table 1]. After a thorough review of the data on animal cloning, the NAS panel, in its report Scientific and Medical Aspects of Human Cloning [page ES-1], came to the following conclusion: "It [cloning-to-produce-children] is dangerous and likely to fail."
Stem Cells and Regenerative Medicine
The subject of stem cell research is much too large to be covered extensively here. Yet the following information on stem cells and their possible uses in medical treatments should facilitate understanding of the relationships between cloning-for-biomedical-research and stem cells (see also the reports Scientific and Medical Aspects of Human Reproductive Cloning and Stem Cells and the Future of Regenerative Medicine).
Stem cells are undifferentiated multipotent precursor cells that are capable both of perpetuating themselves as stem cells and of undergoing differentiation into one or more specialized types of cells (for example, kidney, muscle). Human embryonic stem cells have been isolated from embryos at the blastocyst stage15 or from the germinal tissue of fetuses.16 Multipotent adult progenitor cells have been isolated from sources such as human17 and rodent18 bone marrow. Such cell populations can be differentiated in vitro into a number of different cell types, and thus are the subject of much current research into their possible uses in regenerative medicine. Cloned human embryonic stem cell preparations could be produced using somatic cell nuclear transfer to produce a cloned human embryo, and then taking it apart at the (100-200 cell) blastocyst stage and isolating stem cells (see Figure 2). These stem cells would be genetically virtually identical to cells from the nucleus donor.
Scientists are pursuing the development of therapies based on transplantation of cells for several human diseases, including Parkinson's disease and Type I diabetes. In Parkinson's disease, particular brain cells that produce the essential neurotransmitter dopamine die selectively. Experimental clinical treatment involving transplantation of human fetal brain cell populations, in which a small fraction of the cells produce dopamine, has improved the condition of some Parkinson's disease patients.19 Dopamine-producing neurons derived from mouse embryonic stem cells have been shown to function in an animal model of Parkinson's disease.20 Thus, there is a possibility that transplantation of dopamine-producing neural cells derived from embryonic or adult stem cell populations might be a useful treatment for Parkinson's disease in the future.
However, to be effective as long-term treatments of Parkinson's disease, Type I diabetes, and other diseases, cell transplantation therapies will have to overcome the immune rejection problem. Cells from one person transplanted into the body of another are usually recognized as foreign and killed by the immune system. If cells derived from stem cell preparations are to be broadly useful in transplantation therapies for human diseases, some way or ways around this problem will have to be found. For example, if the cells were isolated from a cloned human embryo at the blastocyst stage, in which the donor nucleus came from a patient with Parkinson's disease, in theory these stem cells would produce the same proteins as the patient. The hope is that dopamine-producing cells derived from these "individualized" stem cell preparations would not be immunologically rejected upon transplantation back into the Parkinson's disease patient. Alternatively, if dopamine-producing cells could be derived from the patient's own adult stem cell or multipotent adult precursor cell populations, they could also be used in such therapies. Another possibility is mentioned in a press report21 about work with a single Parkinson's disease patient, in which brain cells were removed from the patient, expanded by growth in vitro, stimulated to increase dopamine production, and transplanted back into the brain of the same patient with an observed reduction in disease symptoms.
By combining specific gene modification and cloned stem cell procedures, Rideout et al.22 have provided a remarkable example of how some human genetic diseases might someday be treated. Starting with a mouse strain that was deficient in immune system function because of a gene mutation, these investigators (1) produced a cloned stem cell line carrying the gene mutation, (2) specifically repaired the gene mutation in vitro, (3) differentiated the repaired cloned stem cell preparation in vitro into bone marrow precursor cells, and (4) treated the mutant mice with the repaired bone marrow precursor cells and observed a restoration of immune cell function.
Although remarkably successful, the experimental results included a caveat. The investigators also observed a tendency of even these cloned bone marrow precursor cells to be recognized as foreign by the recipient mice. Rideout et al. were led to conclude: "Our results raise the provocative possibility that even genetically matched cells derived by therapeutic cloning may still face barriers to effective transplantation for some disorders."
Lanza et al.23 have also evaluated the potential for immune rejection of cloned embryonic materials, while showing the potential therapeutic value of tissues taken from cloned fetuses. Cloned cattle embryos at the blastocyst stage were transferred to the uteri of surrogate mothers and allowed to develop for five to eight weeks. Fetal heart, kidney, and skeletal muscle tissues were isolated, and degradable polymer vehicles containing these cloned cells were then transplanted back into the animals that donated the nuclei for cloning. The investigators observed no rejection reaction to the transplanted cloned cells using two different immunological tests. More investigations with cloned stem cell materials involving different stem cell preparations of varying sizes, different sites of implantation, and sensitive tests to detect low levels of immunological rejection will be required for a complete assessment of the possibility of using cloned stem cell populations to solve the immune rejection problem.
[Modified from Rick Weiss and Patterson Clark, The Washington Post.]
Producing cloned stem cell preparations for possible use in individual patients suffering from diseases like Parkinson's disease and Type I diabetes is one reason to pursue cloning-for-biomedical-research.24 In vitro production of cloned human embryos could also be important to scientists interested in studying early human development. Stem cells derived from cloned human embryos at the blastocyst stage that were produced with nuclei from individuals with genetic diseases could be useful in the study of the critical events that lead to these diseases (for example, see Bahn et al.25 ). Specific genes could be introduced into developing human embryos to obtain information about the role or roles of these genes in early human development.
In the experiments described by Cibelli et al., the stated intent was to create cloned human embryos that would progress to the 100-200 cell stage, at which point the cloned embryo would be taken apart, stem cells would be isolated from the inner cell mass, and an attempt would be made to grow and preserve "individualized" human stem cells (see Figure 2) for the possible future medical benefit of the somatic cell donor. Because the cloned human embryos stopped dividing and died at the six-cell stage, no stem cells were isolated in these experiments. In light of results in other animal species and the variable completeness of "epigenetic reprogramming," it is perhaps not surprising that sixteen of the nineteen cloned human embryos described by Cibelli et. al. did not undergo cell division and none of the other three divided beyond the six-cell stage.
Although the steps these researchers followed in these experiments were the same as those that would be used by those attempting human cloning-to-produce-children, they distinguished their intent from such cloning by stating: "Strict guidelines for the conduct of this research have been established by Advanced Cell Technology's independent Ethics Advisory Board (EAB). In order to prevent any possibility of reproductive cloning, the EAB requires careful accounting of all eggs and embryos used in the research. No embryo created by means of NT [nuclear transfer] technology may be maintained beyond 14 days of development."
Parthenogenesis (Another Form of Asexual Reproduction)
Using chemical or electrical stimuli, it is also possible to stimulate human eggs to undergo several rounds of cell division, as if they had been fertilized (see Figure 1). In this case, the egg retains all forty-six egg cell chromosomes and egg cell mitochondria. In amphibians, this asexual reproduction process, known as parthenogenesis, has produced live offspring that contain the same nuclear DNA as the egg. These offspring are all necessarily female. Parthenogenesis in mammals has not led reproducibly to the production of live offspring. 27
Cibelli et al.26 activated human eggs (obtained from informed and consenting donors) by parthenogenesis, and obtained multiple cell divisions up to the early embryo stage in six out of twenty-two attempts. Although there was no report that stem cells were isolated in these experiments, it is possible that parthenogenesis of human eggs could induce them to develop to a stage where parthenogenetic stem cells could be isolated. For example, Cibelli et al.28 derived a monkey parthenogenetic stem cell preparation from Macaca fasicularis eggs activated by parthenogenesis. Whether cloned stem cells resulting from parthenogenesis have been completely and correctly epigenetically reprogrammed remains to be determined.