Pathways to Pluripotency: How Germ Cells Make Stem Cells - Male Germline Stem Cells Developmental and Regenerative Potential - page 11

integrin and C-Kit in the developing embryo (Morita-Fujimura et al. 2009 ), indicating
there is likely to be heterogeneity within the starting population of PGCs used to
derive EG cells reflecting heterogeneity present in vivo . Cell sorting experiments also
found PGCs expressing low or no a6 integrin were more able to give rise to EG cells
(Matsui and Tokitake 2009 ). In addition, these studies examined evidence for the
presence of a side population within the germ cell pool. When cells are stained with
Hoechst 33342 dye and analyzed by flow cytometry using a UV laser it is possible to
identify differently stained populations depending on the ability of the cells to dis-
charge the Hoechst dye. Cells that express high levels of the ATP binding cassette
reporter ABCG2 discharge the dye and therefore are less strongly stained by the dye.
These cells constitute a distinct population of cells, the so-called side population.
Interestingly, a large fraction of the PGC pool at 10.5 dpc can be defined as side popu-
lation cells and moreover these cells showed an enhanced ability to give rise to EG
cells (Matsui and Tokitake 2009 ). The implications of these results remain to be
determined but the identification of some markers of PGC heterogeneity provides a
powerful handle with which to determine the underlying molecular mechanisms.
The analysis of how PGCs can be converted into EG cells has been studied to
some extent (Fig. 1.3 ). An important first question is how growth factors act on the
PGCs in the first place. Compelling evidence based in part on genetic studies
strongly suggests that KL acts directly on the PGCs via the C-Kit receptor tyrosine
kinase (Dolci et al. 1991 ; Godin et al. 1991 ; Matsui et al. 1991 ). Similar but less
compelling data suggest that FGF and LIF also act directly on the PGCs via specific
receptors or receptor complexes (Resnick et al. 1998 ; Cheng et al. 1994 ; Takeuchi
et al. 2005 ; Durcova-Hills et al. 2006 ). Several studies suggest that PGCs express
FGF receptors during the period in which they are susceptible to conversion into
EG cells (Resnick et al. 1998 ; Takeuchi et al. 2005 ; Durcova-Hills et al. 2006 ).
However, the methods for culturing PGCs and inducing them to form EG cells
involve the use of fibroblast feeder cells that themselves could respond to FGFs,
and, in general, the isolated PGCs are contaminated with large numbers of embry-
onic somatic cells. So it remains formally possible that FGFs act indirectly to effect
PGC conversion into EG cells. Derivation of EG cells from PGCs that have been
separated from embryonic somatic cells by cell sorting rules out a major contribu-
tion by contaminating embryonic somatic cells in the process (Matsui and Tokitake
2009 ). Interesting studies by Durcova-Hills and colleagues have also shown that in
the conditions used in their studies, FGFs produced by the feeder cells are unlikely
to be important in EG derivation and that up-regulation of FGF2 within the PGCs
themselves may be a key event (Durcova-Hills et al. 2006 ). Further, these studies
suggest that FGFR3 activation within the PGC pool may be critical for conversion
into EG cells as they observed up-regulated expression of FGFR3 within some
PGCs (Durcova-Hills et al. 2006 ). In addition it was noted that conversion of PGCs
into EG cells is associated with altered localization of the FGFR3 receptor from the
cell surface to the nucleus. Taken together these studies suggest that FGFs act
directly on PGCs to effect their conversion to EG cells and do so by activation of
an FGF receptor, specifically FGFR3. More recent studies have shown that tricho-
statin A (TSA), a histone deacetylase (HDAC) inhibitor, can replace FGF2 in the