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alternative methods have been devised to “reprogram” differentiated cell
toward pluripotency; nuclear transfer (NT), fusion with a pluripotent stem
cell partner, and epigenetic reprogramming using defined transcription fac-
tors or microRNAs (reviewed in Yamanaka & Blau, 2010 ).
In NT experiments, differentiated nuclei are reprogrammed by injecting
them into the specialized environment of an enucleated oocyte or a fertilized
egg. This method was initially developed in amphibians and was used to
demonstrate nuclear equivalence; showing that the nucleus of a differenti-
ated cell could generate an entire organism after sequential transfer and
conditioning in oocytes ( Briggs & King, 1952; Gurdon, 1962; Gurdon
et al., 1975; King & Briggs, 1955 ). Three decades later, the first successfully
cloned mammal, Dolly the sheep, was obtained by a similar approach
( Wilmut, Schnieke, McWhir, Kind, & Campbell, 1997 ). Since then, clon-
ing in a variety of species has been reported, although the method remains
labor intensive and intrinsically inefficient as most cloned embryos fail to
complete gestation (reviewed in Yamanaka, 2008 ).
A second approach for reprogramming is achieved by fusing a differen-
tiated cell with a pluripotent cell line. Various stem cell partners have been
used including ESCs, embryonic germ cells, and embryonic carcinoma cells
( Miller & Ruddle, 1976; Tada, Tada, Lefebvre, Barton, & Surani, 1997;
Tada, Takahama, Abe, Nakatsuji, & Tada, 2001 ). In the resulting hybrid,
the somatic genome expresses pluripotency-associated markers (such as
Oct3/4), acquires an ESC-like epigenetic state, and adopts a DNA methyl-
ation and histone modification pattern reminiscent of ESCs ( Kimura, Tada,
Nakatsuji, & Tada, 2004 ). Transplantation of these tetraploid cells into nude
mice results in the formation of teratomas comprising tissues from all three
germ layers ( Tada et al., 2001 ). These data are consistent with the original
differentiated cell being reprogrammed following fusion to become plurip-
otent. Reprogramming using human ESCs as the fusion partner has also
been demonstrated ( Cowan, Atienza, Melton, & Eggan, 2005; Yu,
Vodyanik, He, Slukvin, & Thomson, 2006 ). Although the pluripotent tet-
raploids generated by this approach are not suitable for clinical use, the
methodology does provide some unique perspectives for those interested
in investigating the molecular mechanisms that underlie reprogramming.
Following cell-to-cell fusion, the partner nuclei remain separate for
the first 3 days during which reprogramming is initiated. Subsequently, the
participating nuclei fuse to generate hybrid cells in which the chromosome
content is doubled ( Pereira et al., 2008 ). As the expression of pluripotency-
associated genes begins before the nuclei fuse, this gives experimentalists
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