Conclusion

Cloned animals that are born and survive beyond the neonatal period and appear generally of good health seem to be capable of lifespans that are normal for the species. Dolly was born in mid-1996 and died in 2003. A majority of cloned mice of otherwise apparently normal phenotypes display normal lifespans (Yanagimachi, 2002). These results illustrate that reproductive cloning is possible, without profound restrictions on lifespan. Serial NT studies further illustrate the ability of SCNT to drive somatic cell genomes to extended replicative potential. It seems clear, therefore, that SCNT methods can expand the proliferative lifespan of the donor cell genome. The degree to which this occurs, however, appears to be affected by such parameters as the donor age, the donor cell type, and the differentiated state of the donor cell. In a broader context, SCNT may provide the means necessary for preserving individual lifespan as well.

The potential for SCNT to be employed to derive ESCs for therapeutic purposes has been demonstrated in both animal models and in humans (Rideout et al., 2002; Hwang et al., 2004; review, Latham, 2004). Additionally, the ability of cloned ESCs to rescue phenotypic defects has been shown in mice, effectively illustrating the principle of therapeutic cloning (Rideout et al., 2002). In moving forward with devising strategies for therapeutic cloning, several pertinent questions arise. One is the issue of mutation load. It may be advantageous to restrict donor cell types to those that may experience the least risk of somatic mutation or aneuploidy. Of course, two kinds of mutations must be considered: mutations that affect donor cell phenotype, and thus may be detectable using methods that could permit the exclusion of mutant cells, and "silent" or "masked" mutations that do not immediately affect the donor cell phenotype because they arise in unexpressed genes, or because additional mutations would be needed to allow penetrance. In the latter case, ESCs derived by therapeutic cloning might bear some measurable increase in risk of cancer development if they harbor oncogenic mutations. Selection for stem cells from adult tissues may be worth considering, because stem cells may have less risk of accumulating genetic mutations, as a result of active DNA replication and associated expression of DNA repair proteins, selective partitioning of newly replicated DNA strands (Potten et al., 2002; Merok et al., 2002), and apoptosis of cells with damaged DNA. Additionally, stem cells may confer advantages with respect to mitochondrial phenotype and telomerase expression.

Another consideration is whether the age of the donor individual/patient affects the likely outcome of therapeutic cloning. Will cells from older versus younger individuals display differences in the quality of ESCs that can be produced? Might there arise analytical methods to enrich for the appropriate population of cells, making it possible to avoid such an age effect? Might cells from older individuals require serial NT procedures in order to eliminate defective mitochondria or to achieve suitable telomere lengths? Will younger recipients/patients require higher quality or "younger equivalent'' ESCs to be produced, with respect to mitochondria or telomere lengths?

Epigenetic changes may also be problematic, as cloned embryos often display defects in DNA methylation and regulation of imprinted genes (Latham, 2004). During cloning in animals, clones with severe epigenetic changes are likely to die during gestation, and thus be eliminated.

Cloning to produce ESCs, however, would not require this level of selection, because the embryos are converted to ESCs before this is likely to happen. It may thus be necessary to characterize ESCs thoroughly with respect to methylation state and expression of imprinted genes before using in therapeutic applications.

Given that cloning has been successful in so many different mammalian species, it seems likely that therapeutic cloning should be feasible in humans. However, recent studies indicate that cloning may be more difficult in primates and may not be possible using the protocols described to date. Cloned rhesus monkeys were produced using blastomere nuclei (Meng et al., 1997), and an additional advanced pregnancy was again achieved later (Mitalipov et al., 2002). Cloned rhesus monkeys have not been produced successfully using adult or fetal somatic cell nuclei (Mitalipov et al., 2002; Simerly et al., 2003). It appears that this limitation may reflect an inability of adult somatic cell components, in particular the centrosome, to substitute for authentic sperm-derived components, as indicated in studies by Simerly et al. (2003, 2004) and Miyara et al. (2006). This deficiency could lead to incorrect mitotic segregation of chromosomes and aneuploidy, which would account for the failure to obtain live progeny. The use of cloned human embryos to produce ESCs (Hwang et al., 2003) was clearly a milestone with respect to illustrating the feasibility of the approach. Further research will be needed to identify, and hopefully correct, potential pitfalls in applying this technology in medicine.

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