Appendix N. 
401 
function normally after transplantation into an animal. Possible 
explanations include (i) that epigenetic instability in ES cells is a 
consequence of propagation of cells in tissue culture or (ii) that 
epigenetic instability is a prerequisite for cells to be pluripotent, i.e., 
this instability may be a manifestation of a plasticity in the gene 
expression program that is required to enable the ES cells to 
generate a wide variety of differentiated cell lineages. 
Whatever the explanation for the observed epigenetic instability 
of ES cells may be, it supports the view that the process of 
generating ES cells erases all epigenetic memory of the donor 
nucleus and, as a consequence of the selection process, generates 
epigenetic instability in the selected cells. In other words, epigenetic 
instability appears to be an intrinsic characteristic of ES cells 
regardless of whether derived by SCNT or horn a fertilized egg. This 
is consistent with the conclusion that both types of ES cells have an 
equivalent potency to generate functional cells in culture and, in the 
longer term, fully normal differentiated tissues upon implantation of 
these cells in vivo. 
(iv) ES cells form normal chimeras but abnormal nuclear clones 
As outlined above, faulty reprogramming leads to abnormal 
phenotypes of cloned mice derived from ES cell donor nuclei. Why is 
faulty reprogramming and epigenetic instability a problem for 
reproductive cloning but not for therapeutic applications? The main 
reason for this seeming paradox is that, in contrast to reproductive 
cloning, the therapeutic application of NT does not require the 
formation of a fetus. Therapeutic applications involve the ability of 
cloned ES cells to form a single tissue or organ, not to recapitulate all 
of fetal development. For example, normal fetal development requires 
faithful expression of the imprinted genes. As outlined above, nuclear 
cloning causes between 30% and 50% of imprinted genes to be 
dysregulated consistent with the notion that disturbed imprinting is 
a major contributing factor to clone failure. As most imprinted genes 
have no known function in the postnatal animal, the dysregulation of 
imprinting would not be expected to impede functionality of in vitro 
differentiated ES cells because this process does not require the 
formation of a fetus. Therefore, the functionality of mature cells 
derived in culture from ES cells would not depend on the faithful 
reprogramming of the imprinted genes. Dysregulation of some 
imprinted genes such as Igf2 are known, however, to cause disease 
in the adult. Thus, it will be important to test whether dysregulation 
of such genes has adverse effects on the function of somatic cells 
derived from ES cells. 
PRE -PUBLICATION VERSION 
