Download TGFβ in the differentiation of embryonic stem cells

Cardiovascular Research 74 (2007) 256 – 261
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Review
TGFβ in the differentiation of embryonic stem cells
Michel Pucéat ⁎
INSERM U421/I-Stem, 1 rue de l'internationale, Evry, 91004, France
Received 31 August 2006; received in revised form 7 December 2006; accepted 13 December 2006
Available online 16 December 2006
Time for primary review 24 days
Abstract
The biology of embryonic stem (ES) cell lines has opened new avenues both in the biology of pathophysiological development and in
potential regenerative medicine. The transforming growth factor (TGF)-β superfamily plays a major role in the development of organisms.
The family comprises a variety of growth factors that feature disparate functions in the biology of ES cells. These factors regulate both
stemness and various cell differentiation pathways. Despite intensive work, the role of this family of growth factors in the function of ES cells
is still unclear. More specifically, mouse and human ES cells differentially respond to these factors. Inspired by the biology of development,
this review summarizes the current knowledge on the pleiotropic effects of these growth factors on the fate of ES cells.
© 2006 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
Keywords: Embryonic stem cells; Cardiomyocytes; Growth differentiation factors
1. Introduction
Embryonic stem (ES) cells are derived from blastocysts,
multicellular structures originating from four (human) to five
(mouse) cleavages of fertilized oocytes. Isolated from the
inner cell mass of blastocysts, the ES cells retain properties of
self-renewal and the potential to be committed and to
differentiate toward most cell lineages. They are thus able
to spontaneously give rise to different progenies of the three
embryonic layers, namely, the ectoderm, the mesoderm and
the endoderm. Derivation of embryonic stem cell lines has
opened new avenues both in biology of pathophysiological
development and in potential regenerative medicine.
The transforming growth factor (TGFβ) superfamily plays
a major role in the biology of development. TGFβ and
associated members of the family are mostly pleiotropic
growth factors. They are broadly expressed throughout the
body and regulate many cellular pathophysiological processes
including cell fate, cell proliferation, cell senescence, and
tissue repair. Accordingly, these growth factors have been
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E-mail address: [email protected].
widely investigated in the biology of ES cells. Inspired by
developmental biology, this review summarizes the current
knowledge on the role of TGFβ superfamily members in both
self-renewal and lineage commitment of mouse and human ES
cells. It further explores the intracellular mechanisms
underlying the action of TGFβ-related factors.
2. Intracellular signalling pathways of the TGFβ
superfamily
To better understand the function of TGFβ superfamily
members, their intracellular signalling pathways have to be
well defined. Indeed, recent data about specificity of functions
of Smads [1–4], which constitute the canonical signalling
pathway of TGFβ superfamily, revealed a fine tuning regulation of this pathway and, in turn, of downstream transcriptional
networks.
The family of TGFβ first discovered 25 years ago includes
about forty polypeptidic growth factors that share similarities in their structure. It is classified into two groups: the first
group includes the bone morphogenetic proteins (BMP) and
the growth differentiation factors (GDF) and the second one
comprises TGFβ, activin, and nodal. Three isoforms of TGFβ,
0008-6363/$ - see front matter © 2006 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.cardiores.2006.12.012
M. Pucéat / Cardiovascular Research 74 (2007) 256–261
TGFβ1, 2 and 3, and at least 20 BMPs and 15 GDFs have been
described thus far. All these factors, whose expression and
function have been conserved in many species throughout
evolution, play a key role during embryonic development, in
processes of cell differentiation [5].
The signalling pathways underlying the cellular function of
the TGFβ superfamily members are several: Smad factors are
the key mediators of the canonical TGFβ pathway. The TGFβ
superfamily members bind to receptor complexes composed
by TGFβ receptor type I, activin receptor I, and receptors type
II (TGFβRII, BMP-R II, activin-RII), also called activin-like
kinase receptor (Alk). These receptors feature two transmembrane glycoproteic domains which are able to dimerize through cystein residues following binding of a ligand. Upon
agonist binding, the change in conformation of receptors induces activation of the serine/threonine kinase of RII that phosphorylates RI on specific serine and threonine residues present
in the juxtamembrane glycine and serine-rich GS domain [6].
Phosphorylation-dependent activation of intracellular cytosolic mediators called Smads thus follows and leads to their nuclear translocation. This results in transcriptional activation
and expression of target genes. This signalling pathway is
regulated by cross-talks with other pathways including Wnt,
Hedgehog or other tyrosine kinases-linked growth receptor
signalling pathways. The intracellular signalling is tightly
dependent upon the cell type or stage of cell differentiation.
Smads are divided in two subgroups: the regulatory Smads 2/
3, downstream of TGFβ-R, and 1,5,8, downstream of BMP,
activin, and nodal receptors, and the integrative Smad4 which
is the only one to bind DNA [6]. Smads feature a high
specificity as to their targets and they work in a combinatorial
manner [1–4]. This provides the cell with both a high specificity and a broad diversity in regulation of downstream
transcriptional pathways. Smads include both NLS and NES
sequences which allow these factors for shuttling between the
cytosol and the nucleus. However, DNA binding of Smad4 is
weak and requires co-factors (i.e. transcription factors) that
also bind DNA to turn on gene transcription.
TGFβ superfamily members also activate MAPKs.
Stimulation of TGFβ receptors turns on the activity of the
mitogen-activated protein kinase kinase kinase (MAPKKK)
TAK, acting via the p38 MAP kinase on the transcription
factors CREB and ATF2. This pathway regulates many
cellular processes including cardiac cell hypertrophy [7]. It is
however unclear what the TGFβ-dependent MAPK signalling pathway regulates in ES cells. Ying et al. did not find any
effect of BMP on p38 activation in mouse ES cells [8]. We
also did not observe any effect of p38 or ERK inhibitors on
the TGFβ signalling pathway in mouse ES cells [9].
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growth factors on cell differentiation. This research has thus
been inspired by the biology of mouse development. Specifically, the heart is one of the first organs to form during
embryogenesis. Myocardial precursors are present as early as
in the posterior lateral region of the epiblast. They still
express the POU transcription factor Oct-3/4 at early
gastrulation [10], expression that predicts a role of this so
called ES cell-specific transcription factor, in early mesodermal cardiogenic fate [9]. Then, at gastrulation, following
ingression of the epiblast at the primitive streak, cardiac
progenitors migrate from the anterior region of the primitive
streak into the mesoderm. The first cells to ingress are mainly
those of the heart. In fact, in the mesoderm, the spatial
distribution of cardiac progenitors is mirrored by their
location in the primitive streak. This suggests that the
specification of epiblast cells to the mesodermal lineage is
accomplished through a global control of the timing and
pattern of morphogenetic movement in the course of
gastrulation [10]. The whole morphogenetic process is
under the control of growth factors, including those of the
TGFβ superfamily.
Members of the TGFβ superfamily have been involved in
induction of the mesoderm in Xenopus, zebrafish, chicken
and mouse [11]. Dunn et al. [12] reported that loss of Smad3,
an intracellular component of the TGFβ signalling pathway,
impaired production of anterior axial mesendoderm, while
selective ablation of both Smad2 and Smad3 from the
epiblast disrupts specification of axial and paraxial mesodermal derivatives. The same authors further observed that
Smad2/Smad3 double homozygous mutants lacked mesoderm. Thus, they demonstrated that dose-dependent Smad2
and Smad3 signals cooperatively mediate mesodermal
specification in the early mouse embryo. Besides a direct
regulation of specification of mesoderm, TGFβ potentiates
FGF activity [13]. TGFβ is expressed early in the cardiac
region of the mesoderm. BMPs also feature an important
function in cardiogenesis. BMP-2 is expressed in the early
embryo: added to the culture medium of embryos in vitro,
the morphogen induces differentiation of the anterior median
mesoderm between days 5 and 7 although this region is not a
cardiogenic one [14]. In line with these data, BMP2 triggers
ectopic expression of Nkx2.5, GATA-4, -5, and -6 [14].
BMP4 knockout mice fail to gastrulate and do not form
mesoderm. BMPR1 and 2b receptor-deficient embryos also
fail to form mesoderm [15].
These findings point out the crucial role of TGFβ
superfamily members in directing a mesodermal fate during
early embryogenesis. The biology of mouse development
provided a rationale for investigating the function of TGFβ
superfamily in early specification of ES cells.
3. Short overview of early cardiac development and role
of the TGFβ superfamily: a model to direct the fate of
ES cells
4. Role of TGFβ superfamily in self-renewal of
embryonic stem cells
ES cells recapitulate the early stages of development.
They represent an appropriate model to study the function of
Members of the TGFβ superfamily have been reported to
play a role in both maintenance of self-renewal and lineage
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M. Pucéat / Cardiovascular Research 74 (2007) 256–261
commitment of ES cells although their function is disparate.
Indeed, the effect of TGFβ in stem cell renewal and lineage
specification depends upon species, the cell line, and the
timing. In mouse ES cells, BMP4, through Id proteins, but
not TGFβ, was shown to sustain self-renewal in combination
with LIF [8], in the absence of feeder cells or serum. This
finding, if validated in human ES cells, is of importance to
set up a culture of feeder-free ES cells for future clinical use.
The effect of BMP looks specific to BMP/Smad1/5 although
the reason for that specificity is unclear since TGFβ/Smad
signalling is functional in both mouse [9] and human ES
cells [16]. Nodal [17] and TGFβ [18] might also play a role
in maintenance of human ES cell pluripotency when added at
low concentration in the culture medium. The authors
discussed that nodal might exert its effect by blocking
neuroectodermal differentiation, a default differentiation
pathway of ES cells. Nodal expression is maintained in
human ES cells by Smad2/3-mediated activin signalling.
FoxH1, a target of Smad2/3, regulates transcription of nodal
and its antagonists Lefty A and B [16]. Recently, Xiao et al.
reported that activin A (10 ng/ml) was necessary and
sufficient to maintain pluripotency of HES cells H1 and I6
cultured on matrigel. Activin exerts its action through
inhibition of BMP signalling, which would rather promote
differentiation of cells [19].
Yao et al. [20] also reported that activin prevented
differentiation of HESC toward the trophoectoderm under
feeder-free conditions and in a chemically defined medium.
Altogether, these findings raise many questions as to the
genuine function of the TGFβ superfamily in ES cell
renewal. As with any pleiotropic factor, the effects of
TGFβ superfamily members depend on their concentration
as well as upon the presence of other factors. The identity of
the latter is nevertheless difficult to uncover when ES cells are
cultured on feeder cells, matrigel, and/or in medium
containing serum or a serum replacement. Interestingly, a
pioneering study by Yao et al. [20] showed that addition of
BMP4 to their chemically defined medium containing activin
for a long term (8–10 days) induces expression of cardiac
markers in HESC cultured without feeders. This suggests that
the combination of BMP and activin might constitute a
cardiac induction cocktail. However this medium includes
B27 medium, which contains bovine serum albumin that
might trap many growth factors interfering with BMP
signalling. More experiments performed under better defined
and standardized conditions are still required to assess
whether the differential effects of TGFβ superfamily
members in mouse and human ES cells are only due to the
species and not to the different culture conditions used.
Furthermore, by recruiting different numbers of receptors,
different concentrations of factors are likely to determine the
strength of the intracellular cascade. This might lead to
activation of specific transcriptional targets and, in turn,
might affect specific cell functions. It is also worth pointing
out that most of the experiments published thus far have been
performed with a specific human ES cells line. Very few
reports have compared the results obtained in different cell
lines. As an example, nodal, which in combination with FGF
can allow for the growth of the H9 cell line in the absence of
feeder cells, is not able to support the propagation of HUES1, -3, or -9 cell lines in the absence of feeder cells. Rather, it
induces expression of mesodermal genes (our unpublished
data) as recently reported [21].
5. Role of TGFβ superfamily in embryonic stem cells
differentiation
5.1. Cardiac differentiation
Nodal, TGFβ, and BMP2 are all able to trigger expression
of mesodermal (Brachyury, Tbx6…) and cardiac (Nkx2.5,
Mef2c…) specific genes in mouse ES cells [22,9,23]. BMP4
also favours mesodermal specification of ES cells by
upregulating brachyury in a concentration-dependent manner. It concomitantly blocks neuroectodermal differentiation
[15]. BMP2-induced mesodermal and cardiac specification
is translated into a full cardiogenic differentiation program
leading to an enrichment of cardiomyocytes within embryoid
bodies [22], a three dimensional structure including the three
embryonic layers and recapitulating the early steps of
embryogenesis. BMP2 switches on mesodermal (Tbx6)
and cardiac (Nkx2.5) specific genes via Smad transcriptional
activation [24,25]. Nanog, an ES cell-specific transcription
factor underlying their pluripotency, blocks this signalling
pathway by binding Smad1 [26].
The BMP-dependent cardiogenic effect was observed
only if TGFβ or BMP2 was added to ES cells prior to
differentiation or during the first two days of EB formation.
The opposite effect was revealed if the factors were added
later on to differentiating EBs [22]. These data emphasise the
crucial importance of the time window of addition of the
factor to obtain a specific cellular effect. Interestingly,
expression and associated function of a factor in ES cells and
derivatives often reflects the scenario that occurs in the
embryo. As an example, the EGF–CFC molecule cripto, a
partner in nodal signalling is first expressed in vivo in the
inner cell mass of the blastocyst. Later on, it is found at E6.5
in the epiblast and in the primitive streak, in the mesoderm,
and then at E8.5 in the cardiac region [27]. Cripto is also
essential for cardiogenesis in EBs [28,29] and it turns out
that cripto first expressed in ES cells but no longer in ES cellderived cardiomyocytes is essential for cardiogenesis at a
precise timing. Cripto acts mainly but not only by recruiting
nodal at ALK 4 or 7 receptors and by further activating
Smad2/3. The intracellular mediators, in combination with
co-factors then switch on transcription of cardiac specific
genes.
The first evidence for a role of both activin and TGFβ, in
mesodermal commitment of human ES cells, was provided
by Schuldiner et al. [30]. In contrast, Pera et al. [31] reported
that BMP2 added at a concentration of 25 ng/ml to human ES
cells gives rise to extra-embryonic endoderm, while Xu [32]
M. Pucéat / Cardiovascular Research 74 (2007) 256–261
showed that BMP2, 4, and 7 used at high concentration (100
to 300 ng/ml) rather induces differentiation of these cells
toward throphoblasts. These findings reveal how difficult it is
to assess the true effect of these factors when added in the
presence of serum and FGF2 or in the presence of feeder cells
that release many still unknown factors. A recent report, for
example, showed that FGF2 used to support the propagation
of HESC acts on feeder cells (mouse embryonic fibroblasts),
switching on expression of members of the TGFβ superfamily such as BMP4, TGFβ1, Grem1, or Inhba [33]. In the
absence of serum and FGF signalling, BMP2 used at a low
concentration (5–10 ng/ml) strongly induced expression of
mesodermal and cardiac-specific genes in both HES-1 and 3
(D. Melton cell lines) as well as in I6 (J. Itskovitz cell line)
human cell lines both in undifferentiated ES cells and further
in early EBs (manuscript in preparation). The effect of TGFβ
superfamily members may however depend on the cell lines
and on the timing of addition, which could explain the
discrepancies observed in the literature. We observed, for
example, a much stronger response to TGFβ of mouse BS1
ES cell line than of the CGR8 cell line [9].
TGFβ and related factors (BMP2, nodal) use Oct-4 as a
mediator of their effect to commit mouse ES cells toward a
mesodermal fate. Smad4 binds and transactivates Oct-4
promoter leading to upregulation of the transcription factor
following ES cell stimulation with TGFβ1, BMP2 or nodal.
SiRNA and cDNA antisense transfected into ES cells
prevented both Oct-4 expression and mesodermal and
cardiac specification of ES cells. In fact, in vivo Oct-3/4 is
downregulated in the epiblast of nodal-deficient mice [34]. A
similar downregulation was observed in transgenic mice
lacking Smad2 [35]. This led to the absence of the mesodermal marker Brachyury suggesting that a TGFβ-related
signalling pathway is required to maintain Oct-3/4 expression in the early embryo and that Oct-3/4 is required not only
for self-renewal of cells but also for early mesodermal and
cardiac cell commitment in the embryo [9].
Altogether, these data suggest a highly dynamic role for
BMP in committing the fate of ES cells. The variety of
cellular effect of TGFβ-related factors may depend on the
environment surrounding the cells. The switch from selfrenewal to differentiation upon addition of TGFβ related
factors might be triggered by a combination of other signals
induced by other factors (FGF, VEGF secreted by feeders or
ES cells themselves, antagonists of TGFβ superfamily
members) or cell–cell interactions. Furthermore the strength
(i.e. factor concentration-dependent) of the intracellular
signalling may affect the downstream transcriptional cascades. TGFβ superfamily members are likely to use different
intracellular pathways (Id, Oct-4, Smad, MAPK…) to
mediate different cell functions in ES cells.
GDF3 is one of the members of the TGFβ superfamily. It
features 50% identity with BMP2 and 4. It is mammalian
specific and is expressed as early as in undifferentiated
mouse or human ES cells. Its expression decreases when
cells differentiate. It is in fact an inhibitor of BMP pathway.
259
GDF3 overexpressed in human ES cells contributes to the
maintenance of stemness while reduced GDF3 in mouse ES
cells in the absence of LIF prevent their normal differentiation. This highlights the fact that BMP is a differentiating
agent in human ES cells while it would rather favour
stemness in mouse ES cells cultured in the absence of LIF
[36]. As found in human ES cells, BMP however remains a
differentiating factor in mouse ES cells cultured in the
presence of LIF to prevent methylation and in turn repression
of Oct-4 promoter, a key element in the cardiogenic effect of
BMP [9].
Lefty is also a component of the TGFβ superfamily
signalling pathway. Lefty is crucial for mesendoderm
development. It acts by antagonising nodal and other factors
of the family including activin and BMP. Lefty exerts its
function by antagonizing the EGF–CFC signalling pathway
[37]. Lefty is regulated by Smad and is induced upon
differentiation of ES cells. This factor is at the crossroad of
stemness and differentiation process [38]. Dvash et al. [21]
reported recently that it was transiently expressed in
differentiating human ES cells which do not express Oct-4
any longer. They confirmed that lefty expression was regulated
by nodal. Lefty turns out to be a very interesting factor whose
expression might mark out early mesodermal progenitors.
Thus, it still deserves more investigative work to better define
its function in ES cell fate.
5.2. TGFβ and other cell fates
The TGFβ superfamily members are pleiotropic factors.
Furthermore, the combination of TGFβ superfamily members
with other growth factors affects their cellular effects. Besides
cardiac lineage, these factors direct the fate of ES cells towards
multiple cell lineages of the three embryonic layers although
their functions are different in a murine or human context.
A few reports published these last years have revealed a
function of TGFβ in directing the fate of mouse stem cells
towards specific mesodermal lineages. It was first reported
that TGFβ plays a role in smooth muscle differentiation of
ES cells. Using a truncated TGFβRII as a dominant negative
mutant expressed in mouse ES cells, Sinha et al. found that
smooth muscle cell differentiation of ES cells requires
endogenous TGFβ [39]. Differentiation of another cell
component of vessels is regulated by TGFβ. The latent
TGFβ1 binding protein (LTBP)-1 as well as TGFβ1 were
both found as inducers of endothelial cell fate in ES cells
[40]. Antibodies against LTBP-1 prevent endothelial cell
formation within EBs. Similarly, antibodies raised against
TGFβ added to EBs exert the same action while addition of
TGFβ at low concentrations (1 ng/ml) to EBs improves
endothelial differentiation.
BMP4 treatment of EBs induces expression of the
erythroid markers EKFL and GATA1. BMP4 is however
efficient only until day 4 of differentiation [41].
Endodermal lineages derived from mouse ES cells are
also targets of the TGFβ superfamily. Specifically, TGFβ2 is
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M. Pucéat / Cardiovascular Research 74 (2007) 256–261
family is and how they exert their action under controlled
experimental conditions. The transcriptional cascade(s)
switched on by Smad-specific signalling pathways is still
missing in human ES cells. Uncovering the Smad cofactors as
well as the target genes required to direct the fate of ES cells
will advance the field of developmental cognitive research
and of more clinically oriented research in human ES cells
derived from genetically affected embryos.
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Fig. 1. Summary of the effects of the TGFβ superfamily in mouse and
human ES cells. The size of the letters of factors is indicative of the different
concentrations used to obtain a specific cell lineage.
capable of mimicking the effects of pancreatic rudiments and
this effect was enhanced by cmix (a chick putative endoderm
inducer gene) expression to trigger expression of the
endodermal marker pdx1 [42]. TGFβ together with
Hedgehog, retinoid acid, and FGF promote expression of
pancreatic transcription factors in ES cell-derived embryoid
bodies (EB) [43]. Overexpression of nodal in mouse ES cells
induces upregulation of both mesodermal and endodermal
cell markers while it also downregulates neuroectodermal
markers [44].
Interestingly, TGFβ, applied to human ES cell-derived
EBs at different time frames, prevents expression of
endodermal, endothelial and hematopoietic markers, which
contrasts with findings in the mouse in which TGFβ reduced
the level of endodermal markers but increased endothelial
marker expression. The authors interpreted such a difference
between the two species by the different origins of the yolk
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The effects of TGFβ on the hypoblast, from which these cell
lineages are derived in human, would decrease subsequent
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affecting endoderm, would not affect the hemangiogenic
lineages that are epiblast-derived in the mouse [45].
6. Open issues
It is now established that TGFβ superfamily members
participate in cell fate decision of ES cells (Fig. 1). However,
the role of TGFβ in cell cycle of ES cells has been poorly
investigated. We found for example that some canonical
targets of TGFβ such as cdk inhibitors are conserved in ES
cells [9]. Undoubtedly, much more investigative effort has to
be devoted to this important biological question. Recently,
TGFβ has been reported to repress activity of the telomerase
reverse transcriptase [46]; this issue should also be
investigated in ES cells. The family features a variety of
factors and the role of many of them in ES cells stemness or
differentiation has not been investigated yet. It remains to be
established what the specific role of each member of the
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