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|Title: ||Role of Grb2 in growth and differentiation of embryonic stem cells|
|Authors: ||Murray, Helen|
|Supervisor(s): ||Burdon, Tom|
|Issue Date: ||25-Nov-2011|
|Publisher: ||The University of Edinburgh|
|Abstract: ||Embryonic stem (ES) cells are derived from the inner cell mass of the blastocyst
stage embryo. They exhibit unlimited proliferation in culture and have the ability to
differentiate into all three germ layers of the developing organism, a property
defined as pluripotency. Previously it was reported that growth factor-bound protein
2 (Grb2) is required for differentiation of the epiblast, the embryonic tissue that
harbours the pluripotent founder cells of the foetus.
GRB2 is an adapter protein involved in the activation of the mitogen-activated
protein kinase (MAPK) pathway in response to extracellular signals. It has also
been implicated in the activation of the phosphoinositol-3-kinase (PI3K) pathway in
response to fibroblast growth factor (FGF) signaling.
The work presented in this thesis examines the role of Grb2 in ES cells and
describes previously unreported contributions of this adaptor protein in regulating
ES cell growth and differentiation.
It has been previously been shown by others that Grb2 deficient (Grb2-/-) cells grow
relatively normally in ES growth medium containing serum. However, in serum free
conditions (N2B27 medium) in this project, proliferation of Grb2-/- cells is reduced
compared with wild type and “restored” Grb2-/- cells stably expressing a Grb2 cDNA
Under serum free conditions, Grb2-/- cells grow in tight, refractive colonies. Nanog
expression was uniformly upregulated, in contrast to the heterogeneous pattern
reported in serum-based medium. Colony expansion on the substratum appears to
be compromised, although there is no apparent defect in the initial attachment of
Grb2-/- cells. Cell cycle analysis indicates that the slower growth of Grb2-/- cells in
serum free medium could be due to lengthening of the G1 phase of the ES cell
In an attempt to identify the signalling deficiency responsible for the growth defect of
Grb2-/- cells, MAPK activation was restored by two methods, PMA a ligand that
bypasses the requirement for Grb2, and Raf-ER, a conditionally regulated
component of the MAPK pathway that acts downstream of Grb2 in the MAPK
pathway. Although both approaches increased MAPK signalling they were unable to
rescue the growth defect. This suggests that MAPK is not required or alone is not sufficient. Inhibition of Glycogen synthase kinase 3 β (GSK3 β ) is known to augment
growth of ES cells under MAPK inhibition. Surprisingly, GSK3 β inhibition did not
enhance Grb2-/- cell growth. Under GSK3 β inhibition, Grb2-/- ES cells fail to thrive.
It is hypothesised that under these conditions cells undergo hyper-self-renewal at
the cost of growth.
Grb2-/- ES cells are reported to exhibit limited differentiation potential. To examine
the potency of Grb2-/- cells, these cells were subjected to embryoid body (EB) and
monolayer differentiation. Analysis of EBs showed a loss of Gata4, Gata6 and
endoderm marker gene expression. However, markers of ectoderm (Sox1, Pax6,
MAP2), the late epiblast/nascent mesoderm (Brachyury) and markers associated
with gastrulation (Twist and Snail) were expressed. Outgrowths of morphologically
and immunohistochemically identifiable neuronal cells confirmed differentiation of
ectodermal cell types, indicating Grb2 is not required for neuronal differentiation.
However, beating cardiomyocytes could not be identified in Grb2-/- EBs, though
readily found in restored Grb2-/- cells expressing the Grb2 cDNA. This suggests that
there is an essential role for Grb2 in the mesoderm/cardiomyocyte differentiation
pathway. This may be due to a defect in GATA factor expression since these factors
are essential for cardiogenesis.
In serum-free monolayer differentiation, Grb2-/- cells formed neuronal cells.
Additional inhibition of the MAPK pathway using a small chemical inhibitor failed to
prevent this differentiation. However, biochemical analysis of the cells indicates that
this occurs when ERK activation is very low, indicating differentiation was not
Grb2 mediates FGF-MAPK induced exit from the naïve ground state. These data
suggest a Grb2-independent pathway can also facilitate this transition. Grb2 is
dispensable for differentiation in to some lineages. However as differentiation of
Grb2-/- ES cells is restricted, this indicates Grb2 is required for true pluripotency.|
|Sponsor(s): ||Biotechnology and Biological Sciences Research Council (BBSRC)|
|Keywords: ||embryonic stem cell|
|Appears in Collections:||Royal (Dick) School of Veterinary Studies thesis and dissertation collection|
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