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these pathways during reprogramming to EG cells has not been investigated.
Finally, Trichostatin (TSA), a histone deacetylase inhibitor, can replace
bFGF in EG cell derivation (
Durcova-Hills et al., 2008
). However, given
the diverse cellular targets of HDACs including nonhistones (
Bolden,
Peart, & Johnstone, 2006; Drummond et al., 2005
), the exact manner in
which TSA acts during reprogramming is unclear. A major caveat to these
studies is the inability to draw firm mechanistic conclusions in a heteroge-
neous system. The presence of feeders and contaminating somatic cells mean
that any factor added to the culture could be acting directly on PGCs, indi-
rectly on feeders or somatic cells, or any combination of these options
(
Fig. 5.3
). For instance, bFGF has been shown to increase the expression
of both membrane-bound and soluble LIF by feeder cells (
Rathjen et al.,
1990
). The possibility remains that this may create a high local concentration
or that the signal from membrane-bound LIF may be quite different to the
soluble form, as has been demonstrated for SF (
Matsui et al., 1991, 1992
).
Furthermore, RA, FK, and TSA could all be exerting their effects indirectly
through bFGF, by stimulating its release from feeders, somatic cells, or even
PGCs themselves (
Fig. 5.3
). Some factors have been shown to influence sur-
vival or proliferation of PGCs in the absence of feeders including RA
(
Koshimizu et al., 1995
), FK (
de Felici et al., 1993
), LIF (
Koshimizu
et al., 1996
), and bFGF (
Resnick et al., 1998
). However, in these reports,
PGCs are not fully purified and so somatic cells could mediate indirect effects
(
Fig. 5.3
). More accurate assessment of the growth factors required by PGCs,
and for EG cell derivation, requires a system to culture purified PGCs with-
out a feeder layer.
X
Somatic
cells
PGCs
Feeders
Figure 5.3 Schematic of possible interactions in a heterogeneous culture system.
