over 200 oocytes were used in the course of the experiments that generated a single line. However, the scientists made a number of improvements in the procedure as the experiments progressed, increasing the yield of blastocysts and suggesting that the success rate will be improved in the future. This proof of the principles behind generating NT hES cells has made plausible the derivation of more such lines from specifically defined genetic backgrounds.
It is important to note that stem cells made using NT result from an asexual process that does not involve the generation of a novel combination of genes from two “parents.” In this sense, it may be more acceptable to some than the creation of blastocysts for research purposes by IVF (NIH HERP, 1994). It has also been suggested (Hurlbut, 2004) that transfer of genetically altered nuclei incapable of directing full development might make NT acceptable. However, it has been pointed out (Melton et al., 2004) that this approach faces many technical hurdles and does not avoid the need for oocyte donation. At least three methods for generating hES cells from defective embryos have been suggested. One such method involves the use of viable blastomeres extracted from a morula or blastocyst that has been declared dead due to cleavage arrest (Landry and Zucker, 2004). This proposal is untested and is technically challenging. Even if it were possible to identify unequivocally embryos with no chance of further development, the likelihood of then isolating a viable blastomere and generating an ES line is small. There has been only one published report claiming derivation of mES cell lines from isolated 8-cell blastomeres (Delhaise et al., 1996). One cell line was obtained from 52 fully viable, dissociated 8-cell stage morulae.
Two other methods of generating hES cells from defective embryos have been considered: parthenogenesis and androgenesis. In parthenogenesis, an oocyte can be activated to develop without being fertilized by a sperm. The genomic DNA of the resulting embryo is completely maternally derived, which is not compatible with survival to term. Both mouse and nonhuman primate parthenogenetic ES cell lines have been established (Kaufman et al., 1983; Cibelli et al., 2002). The results are of interest because deriving stem cells from parthenogenetic blastocysts could eliminate the requirement to produce and destroy viable blastocysts. Parthenogenetic ES cells could serve as an alternative source for autologous cell therapy. However, parthenogenetic mES cells show restricted tissue contributions in chimeras and in teratomas formed by grafting the cells under the kidney capsule (Allen et al., 1994); this is related to the lack of expression of key imprinted genes that are normally expressed from the paternal genome. In contrast with parthenogenesis, in androgenesis the entire genome comes from the male parent. Such embryos also do not survive to term. Diploid androgenetic mES cells have been derived (Mann et al., 1990), but many androgenetic ES cell chimeras died at early postnatal stages, and the ones that survived developed skeletal abnormalities. Again, the imprinting status of the cells differed from that of wild-type ES cells (Szabo et al, 1994). Thus, although the results show that androgenetic and parthenogenetic ES cells have broad developmental potential, their imprinted gene expression status is likely to