Controlling the genotype of ES cells will also be important in the future if they are to be used directly as therapeutic tools in regenerative medicine. Transplantation of hES cells will face issues of tissue rejection common to all forms of organ or tissue transplants. As in organ or bone marrow transplantation, one solution is to develop large banks of genetically diverse hES cells to increase the chances that matches can be found for all patients who need them. That is one strong medical reason for generating additional hES cell lines from a wider spectrum of the population. Other methods to overcome tissue rejection, including genetic modification of hES cells to reduce immunogenicity and use of immunosuppressive drugs may be helpful. However, in the long run, one obvious solution would be autologous transplantation, using hES cells genetically identical with the recipient of the graft.

Generation of ES cells using nuclear transfer (NT) has the potential to produce ES cells of defined genotype to address both genetic diversity and avoidance of rejection. NT is the process by which the DNA-containing nucleus of any specialized cell (except eggs and sperm, which contain only half the DNA present in other cells) is transferred into an oocyte whose own nuclear genome has been removed (Figure 2.2). The egg can then be activated to develop and will divide to form a blastocyst, whose genetic material and genetically determined traits are identical with those of the donor of the specialized cell, not those of the donor of the oocyte. The oocyte does provide a very small amount of genetic information in the mitochondria, the “energy factories” of the cell, but the genes in the nucleus are of overriding importance, nuclear genes being responsible for the vast majority of the traits of the animal. If such a blastocyst were transferred to a uterus, the transferred blastocyst could potentially develop into a live-born offspring—a clone of the nuclear donor. NT was first developed with frog embryos and later successfully used to generate Dolly the sheep, the first mammal cloned from an adult cell (Campbell et al., 1996). Since the birth of Dolly, live cloned offspring of several other mammalian species have been reported, including mice, goats, pigs, rats, cats, and cows. The success rate of live births is very low, however, and a variety of abnormalities have been found in cloned animals (NRC, 2002b), so this is currently an unreliable technology and unsafe for application to humans. Given the safety issues associated with NT for human reproduction, there is a worldwide consensus that such efforts should be not be conducted at this time. Despite some well-publicized but undocumented claims of production of live cloned babies, the scientific community in general and this committee in particular support that moratorium.

Blastocysts derived using NT can be an important source of genetically defined ES cells. If the inner cell mass of the NT-derived blastocyst, comprising a few dozen undifferentiated cells, is removed and grown in culture, ES cells can be derived and their genotype will be identical with that of the nuclear donor. Successful derivation of pluripotent mES cells from cloned NT blastocysts has been demonstrated in mice by several groups (Kawase et al., 2000; Munsie et al., 2000; Wakayama et al., 2001). In addition, the principle of alleviating a genetic disease was demonstrated by transplantation of genetically repaired mouse NT ES cells in an immunodeficient

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