Fibroblast growth factor (FGF) signalling plays many fundamentally important roles
during the development of the mammalian embryo. However, its effects on
pluripotent stem cells derived from mouse and human embryos appear to be markedly
different. FGF2 is routinely added to culture medium for propagating undifferentiated
human (hES) cells, whereas in mouse (mES) cell cultures FGFs have been described
as regulators of their differentiated progeny. To assess the effect of FGF signalling on
undifferentiated mES cells, the effects of FGF2 and 4 were analysed in the presence
of saturating and sub-saturating levels of the inhibitor of differentiation, leukaemia
inhibitory factor (LIF). Mouse ES cell self-renewal was quantified by measuring the
expression of the stem cell specific reporter Oct4-LacZ in biochemical and
fluorometric assays. Treatment with FGF reduced the expression of the OCT4-LacZ
reporter, even under saturating concentrations of LIF and this was mirrored by
decreased levels of OCT4 protein. Furthermore, treatment with FGF leads to
upregulation of the ectodermal differentiation marker Pax6. These results suggest that
FGF signalling has a direct impact on undifferentiated mES cells, and actively
promotes their differentiation. To asses the effect of FGF signalling on hES cells
without the influence of undefined factors, a feeder and serum free system was
developed. Cells growing in this conditions for >20 passages maintained expression
of surface (SSEA3 and TRA1-60 and 81) and internal (OCT4) markers specific for
undifferentiated hES cells. Expression of these markers was dependant on the
continuous presence of FGF2. Indeed, withdrawal of FGF2 resulted in a rapid
decrease of in hES cell growth and of the emergence of cell flattened morphology and
of the surface marker SSEA1, changes typically associated with differentiation. Two
important signals activated by FGF in hES cells are the ERK/MAPK and PI3K
pathways. To assess their functional relevance, hES cell cultures were treated with
the drugs UO126 and LY294002, inhibitors of the MAPK and PI3K pathways
respectively. Drug mediated suppression of the phosphorylation of these pathways,
correlated with a reduction in cell growth, flattening of the colonies and reduction in
SSEA4 expression. Use of SB431542, specific inhibitor of TGFβ/activin type I
receptor kinase (Alk5) also resulted in the flattening of the colonies and the
appearance of dispersed cells. Therefore, inhibition of MAPK and PI3K appears to
impair growth and self-renewal in hES cells and this may be happening in conjunction
with TGFβ/Activin pathway. Taken together, these results suggest that FGF
signalling has opposite effects in mouse and human ES cells: inducing differentiation
in mES and sustaining self-renewal in hES