HYPOTHESIS
3
Development 138, 3-8 (2011) doi:10.1242/dev.050831
© 2011. Published by The Company of Biologists Ltd
The origin and identity of embryonic stem cells
Jennifer Nichols1,2,* and Austin Smith1,3
Embryonic stem (ES) cells are used extensively in biomedical
research and as a model with which to study early mammalian
development, but their exact origin has been subject to much
debate. They are routinely derived from pre-implantation
embryos, but it has been suggested that the cells that give rise
to ES cells might arise from epiblast cells that are already
predisposed to a primordial germ cell (PGC) fate, which then
progress to ES cell status via the PGC lineage. Based on recent
findings, we propose here that ES cells can be derived directly
from early epiblast cells and that ES cells might arise via two
different routes that are dictated by their culture conditions.
Key words: Cells, ES, Identity
Introduction
Pluripotency is first acquired in the mouse embryo as the epiblast
forms in the inner cell mass (ICM). Under appropriate culture
conditions, ICM cells can proliferate in vitro in the form of
embryonic stem (ES) cells (Evans and Kaufman, 1981; Martin,
1981). These cells retain the capacity to repopulate an embryo and
to contribute descendents to all tissues of the adult, including the
germline (Bradley et al., 1984), and are thus defined as being naïve
pluripotent cells (Nichols and Smith, 2009). Pluripotent stem
cell lines were originally derived from mouse testicular
teratocarcinomas. That these teratocarcinomas arise from germ cells
may be inferred from observations that teratocarcinomas are more
common in the germline than in any other tissue; that testicular
tumours in mice usually arise soon after birth; and that nascent
tumours have been detected specifically within the testicular
tubules of foetuses (Stevens, 1962). Subsequently, it was shown
that pluripotent cell lines that are almost indistinguishable from ES
cells could be derived in vitro from primordial germ cells (PGCs)
of the developing mouse embryo by epigenetic reprogramming
(Matsui et al., 1992). Thus, a window of opportunity during which
to capture naïve pluripotency seems to open twice during
development: first in the early epiblast and later in the germ cell
lineage.
The resemblance of embryonic germ (EG) cells to ES cells
prompted the suggestion that ES cells might arise from epiblast
cells that are already predisposed to a PGC fate (Gardner and
Brook, 1997; Zwaka and Thomson, 2005). It was also speculated
that PGCs might be induced in explants that are cultured from
embryos at pre-implantation or post-implantation stages of
development (Hayashi and Surani, 2009a; Tang et al., 2010). In this
hypothesis article, we propose, based on recent findings, that ES
1
Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Tennis
Court Road, Cambridge CB2 1QR, UK. 2Department of Physiology, Development
and Neuroscience, University of Cambridge, Tennis Court Road, Cambridge
CB2 1QR, UK. 3Department of Biochemistry, University of Cambridge, Tennis Court
Road, Cambridge CB2 1QR, UK.
*Author for correspondence ([email protected])
cells can be derived in explant cultures via two routes: directly
from the newly formed epiblast, as it transits the state of naïve
pluripotency; or during the specification of PGCs in culture when
cells undergo epigenetic reprogramming, resulting in their reacquisition of pluripotency (Fig. 1).
Emergence of naïve pluripotency
Fertilisation in mammals produces a zygote that will give rise to
the entire embryo and to the extra-embryonic lineages. Equipotent
blastomeres are produced from the zygote during several rounds of
cleavage divisions, but by the blastocyst stage, two distinct lineages
have emerged that exhibit morphological and molecular
differences. The trophectoderm, which is characterised by its
epithelial nature and by the expression of the transcription factor
Cdx2 (caudal type homeobox 2), among other specific markers
(Beck et al., 1995), will contribute primarily to the placenta,
whereas the ICM segregates into the epiblast (otherwise known as
the primitive ectoderm) and the hypoblast, which is also commonly
referred to as the primitive endoderm. The epiblast and hypoblast
become completely distinct, at least in the mouse embryo, by the
time of implantation. Each has its own molecular signature.
Notably, the epiblast is characterised by the expression of the
transcription factor Nanog, and the hypoblast by the expression of
the transcription factors Gata6 and Gata4 (GATA-binding proteins
6 and 4) (Fig. 2A) (Chambers et al., 2003; Chazaud et al., 2006;
Plusa et al., 2008). The acquisition of epiblast identity coincides
with the reactivation of the inactive X chromosome in female
mouse embryos (Mak et al., 2004; Okamoto et al., 2004; Silva et
al., 2009). X reactivation is also a hallmark of the successful
formation of ES cells (Silva et al., 2008), and might be one of
several epigenetic events that serve to alleviate chromatin
inaccessibility to enable the establishment of the naïve pluripotent
state in the epiblast. Hypoblast differentiation is not required for
epiblast specification; embryos that carry mutations in genes
encoding components of the fibroblast growth factor (Fgf)/Erk
(Ephb2 – Mouse Genome Informatics) pathway, such as Grb2
(growth factor receptor-bound protein 2), lack hypoblasts but have
apparently normal epiblasts (Chazaud et al., 2006; Cheng et al.,
1998). Chemical inhibition of the Fgf/Erk pathway entirely
suppresses hypoblast development and concomitantly expands the
epiblast (Nichols et al., 2009b; Yamanaka et al., 2010). The epiblast
cells from embryos treated in this way are not compromised in
their developmental potential; they can contribute to normal
embryogenesis and form functional germ cells when injected into
host blastocysts (Nichols et al., 2009b).
Following implantation, the rodent epiblast grows from a ball of
cells into a cup-shaped epithelium. This morphological change is
accompanied by the reduced expression of transcription factors
such as Nanog and Rex1 (zinc-finger protein 42; Zfp42 – Mouse
Genome Informatics) and by the upregulation of Fgf5 and
brachyury (Chambers et al., 2003; Pelton et al., 2002; Thomas and
Beddington, 1996). Additionally, in female embryos, random X
inactivation occurs throughout the epiblast by 5.5 days post coitum
(dpc) (Rastan, 1982). Over the next few days, specification of the
DEVELOPMENT
Summary
4
HYPOTHESIS
Development 138 (1)
A
B
Key
Unspecified early embryonic cells
Hypoblast
Trophectoderm
Early epiblast and ESCs
PGCs and EGCs
germ cell lineage commences in the proximal epiblast, which has
initiated expression of Fgf5 and brachyury. Germ cell precursors
are specified by signalling molecules, such as the bone
morphogenetic proteins (BMPs) Bmp4 and Bmp8b, which are
secreted by the extra-embryonic ectoderm and visceral endoderm,
and are subsequently marked by expression of PGC markers such
as Stella (developmental pluripotency associated 3; Dppa3 –
Mouse Genome Informatics), Blimp1 (PR domain containing 1,
with ZNF domain; Prdm1 – Mouse Genome Informatics) and
Prdm14. Expression of somatic genes such as homeobox A1
(Hoxa1), Hoxb1, Lim1 (LIM homeobox protein 1; Lhx1 – Mouse
Genome Informatics) and Evx1 (even skipped homeotic gene 1
homolog) is suppressed in the Stella-positive cells, but brachyury
and Fgf8 remain expressed in these cells, indicating that they have
been diverted from the naïve pluripotent state of the epiblast and
have embarked upon the acquisition of a somatic fate (Saitou et al.,
2002). PGCs subsequently multiply and migrate along the
developing hindgut to arrive at the genital ridges at around midgestation, maintaining expression of the POU domain transcription
factor Oct4 (Pou5f1 – Mouse Genome Informatics) during this
time. Although Oct4 is considered to be a marker of pluripotency,
PGCs are actually unipotent in vivo; however, they can be induced
to acquire pluripotency when explanted or by transformation into
teratocarcinoma cells.
Derivation of ES cell lines: a brief history
The first ES cells were successfully derived directly from
blastocysts in 1981 (Evans and Kaufman, 1981; Martin, 1981). The
initiative to derive pluripotent cell lines from early embryos was
inspired by studies of teratocarcinoma cells. In addition to their
spontaneous appearance in the testes of mice, these tumours can be
generated by grafting embryos at the egg cylinder stage into an
adult mouse (Damjanov et al., 1971). Undifferentiated embryonal
carcinoma (EC) cell lines can be propagated from explanted
tumours and can contribute to multiple tissues when injected into
host blastocysts to form chimaeras (Brinster, 1974). The culture
regime for retaining pluripotency included a ‘feeder’ layer of
mitotically inactivated mouse fibroblasts and foetal calf serum that
was batch-selected to promote proliferation and retain an
undifferentiated phenotype. ES cells were derived in these
conditions by allowing the blastocysts (or isolated ICMs) to
develop intact for a few days prior to their disaggregation. A
satisfactory outcome of ES cell derivation is confirmed by the
appearance of ES cell colonies after several days of further culture
(see Fig. 2B). For many years, ES cell derivation remained
inconsistent and poorly understood. Interestingly, it became
apparent that the facility for ES cell derivation was dependent upon
the genetic background of the embryo; the 129 strain of mouse was
found to be generally permissive, whereas the derivation of ES
cells from embryos of the CBA strain was not possible without
modification of the original protocol (Batlle-Morera et al., 2008;
Brook and Gardner, 1997; Buehr and Smith, 2003).
Several studies have been performed to try to correlate the
changes that occur during explant outgrowth with the successful
establishment of ES cells. For example, the expression levels of
Oct4, a POU domain transcription factor and marker of pluripotency,
have been shown to decrease more dramatically in outgrowths of
embryos explanted from mouse strains that are resistant to ES cell
DEVELOPMENT
Fig. 1. Derivation of naïve pluripotent cell lines. Derivation of embryonic stem cells (ESCs), embryonic germ cells (EGCs) and primordial germ
cells (PGCs) from mouse embryos under conventional culture conditions. (A)The progression of development in vivo. The founders of naïve
pluripotent cell lines appear as early epiblast (red) in the late blastocyst and as PGCs (yellow) in the embryo at 8.5 dpc. (B)The two proposed
alternative routes to producing naïve pluripotent cell lines from explanted blastocysts in vitro. The outgrowth displaying a red core represents the
early epiblast route of ES cell derivation; the outgrowth with a yellow core represents a longer period of culture during which PGCs may be
specified. Grey arrows represent the alternative speculated pathways from this extended culture to either ESCs or EGCs. The schematic forms are
not to scale.
Fig. 2. Segregation of epiblast and hypoblast in the mouse
embryo. (A)Confocal image of a peri-implantation 4.5 dpc mouse
embryo, immunostained for Nanog (red) and Gata6 (green) to show
segregation of epiblast and hypoblast, respectively. The trophectoderm
is visualised using DAPI (blue). (B)Phase-contrast image of an
embryonic stem cell colony growing on a feeder layer of mitotically
inactivated fibroblasts. Scale bars: 10m.
derivation compared with Oct4 levels in explanted embryos from the
permissive 129 strain (Buehr et al., 2003). Even in 129 embryo
explants, the domain of Oct4 expression has been found to decrease
to a few cells during the outgrowth period, suggesting that successful
ES cell derivation does not simply depend upon the expansion of the
Oct4-positive population of cells, and might actually entail epigenetic
and transcriptional resetting of the expression of key genes, which
possibly creates an artificial, culture-induced state (Buehr and Smith,
2003). As we discuss below, in order to dissect the process of ES cell
derivation and to understand the origin of ES cells, it has been
necessary to refine the crude culture regime for murine ES cell
derivation.
The essential self-renewal function of feeder cells in ES cell
culture protocols can be reproduced by supplementing the culture
medium with the cytokine leukaemia inhibitory factor (LIF), at
least for ES cells derived from embryos of the 129 strain of mice
(Smith et al., 1988; Williams et al., 1988). More recently, serum
has been replaced by the addition of Bmp4 or of related growth
factors to the culture medium, allowing germline-competent ES
cells to be propagated in defined culture conditions by
supplementation with the two cytokines LIF and Bmp4 (Ying et al.,
2003). Unfortunately, even under these defined conditions, it has
not been possible to derive germline-competent ES cells efficiently
from embryos of non-129 strains of mice. This marked variation in
ability to derive ES cells from a range of different mouse strains
might be related to strain-specific variation in Erk signalling
(Batlle-Morera et al., 2008; Wray et al., 2010). The Erk pathway is
activated independently by Fgf4 and LIF (Burdon et al., 1999;
Kunath et al., 2007; Wang et al., 1994), both of which are produced
in early mouse embryos (Nichols et al., 1996; Rappolee et al.,
1994). Erk activity has been associated with differentiation of ES
cells; inhibiting this pathway has been shown to promote ES cell
self-renewal (Burdon et al., 1999). The inhibition of Erk activity
has also improved the efficiency of ES cell derivation from
embryos of non-129 strains of mice (Batlle-Morera et al., 2008;
Buehr and Smith, 2003).
The strain specificity that has hampered ES cell derivation has
now been eliminated by a new approach. Instead of activating
pathways to block differentiation, small molecules are employed to
HYPOTHESIS
5
inhibit specific kinases. This new ES cell culture regime, known as
‘2i’ (for two inhibitors), operates by inhibiting both Erk signalling
and glycogen synthase kinase 3 (Gsk3) (Ying et al., 2008). In
essence, the inhibition of Erk signalling in the developing embryo
abrogates the pro-differentiation activities that are promoted during
normal development (Nichols et al., 2009b). Expansion of ES cell
cultures is inefficient unless GSK3 is inhibited or the Stat3 pathway
is stimulated, but the mechanism for this rescue is not entirely clear
(Ying et al., 2008). It is possible that Gsk3 inhibition promotes
translation in the context of ES cell culture in 2i (Wray et al.,
2010). By using 2i culture conditions, ES cells can be derived
efficiently from all mouse strains tested to date, including the nonobese diabetic (NOD) mouse, previously considered to be the strain
most recalcitrant for ES cell derivation (Hanna et al., 2009;
Kiyonari et al., 2010; Nichols et al., 2009a; Ying et al., 2008). This
important finding demonstrates that a particular inbred genetic
predisposition is not required for ES cell derivation, but that ES
cells arise owing to the generic features of early mouse
development.
Generating pluripotent cell lines from
post-implantation embryos
The generation of EC cells from teratocarcinomas that had
arisen spontaneously in male gonads (Stevens, 1962) prompted
speculation that it may be possible to derive pluripotent stem cell
lines directly from PGCs. This was achieved by culturing the
posterior region of 8.5 dpc mouse embryos on feeder cells in the
presence of LIF, steel factor and Fgf4 (Matsui et al., 1992). These
cell lines are called embryonic germ (EG) cells and they closely
resemble ES cells. It is also possible to derive EG cells from
explanted genital ridges at 11.5 and 12.5 dpc, but by this stage the
developing germ cells have undergone imprinting erasure, which
renders the derivative EG cells less able to integrate into chimaeras,
and particularly into the germline (Labosky et al., 1994; Tada
et al., 1998). The derivation of EG cells involves transient
supplementation with Fgf at explantation, and it has been suggested
that this addition triggered an epigenetic reprogramming process
(Durcova-Hills et al., 2006). However, Fgf appears not to be
necessary if PGCs are cultured directly in 2i medium with LIF
(Leitch et al., 2010). It appears that under these conditions the
epigenetic barrier from PGC to EG cell status may be more easily
traversed. This may be attributable to the capacity of Erk and Gsk3
inhibition to prevent the PGCs from progressing along their normal
differentiation pathway.
The post-implantation mouse epiblast has been shown to give
rise to multi-lineage-competent teratocarcinomas (Diwan and
Stevens, 1976; Solter et al., 1970). However, it has not been
possible to derive ES cells from post-implantation epiblasts
directly. Recently, pluripotent cell lines were generated from postimplantation mouse embryos using the culture conditions
employed for the derivation of stem cell lines from human
blastocysts; specifically, explanting on to a substrate of feeder cells
or fibronectin and supplementation of the medium with activin and
Fgf2 (Brons et al., 2007; Tesar et al., 2007; Thomson et al., 1998).
These ‘EpiSCs’ cannot be propagated efficiently from single cells
and do not readily contribute to chimaeras. They share many
characteristics with the pluripotent lines derived from human
embryos, suggesting that human ‘ES’ cells may actually represent
a more advanced stage of development than the blastocyst (Fig. 3)
(Brons et al., 2007; Nichols and Smith, 2009; Rossant, 2008; Tesar
et al., 2007). Fgf2 is crucial for the derivation and subsequent
expansion of both human ‘ES’ cells and EpiSCs. EpiSCs are
DEVELOPMENT
Development 138 (1)
6
HYPOTHESIS
Development 138 (1)
Fig. 3. Derivation of
pluripotent cell lines from
human embryos. (A,B)A
comparison of pluripotent cell
line derivation protocols for (A)
mouse and (B) human embryos.
This figure highlights the
difference in timing of mouse
and human development in vivo
(in days), and the appearance of
pluripotent cell lines in vitro. It
indicates that the long period of
culture that is required for the
appearance of human
‘embryonic stem’ cells (ESCs)
would allow explanted cells to
progress in vitro to the equivalent
of the post implantation mouse
embryo, from which EpiSCs are
derived.
’
Unspecified early embryonic cells
Hypoblast
Trophectoderm
Early epiblast and ESCs
transcriptionally and epigenetically distinct from ES cells; EpiSCs
are characterised by expression of Fgf5 and brachyury, whereas a
hallmark of ES cells is the expression of Rex1 and Klf4 (Kruppellike factor 4), and the absence of X chromosome inactivation
(Brons et al., 2007; Guo et al., 2009; Tesar et al., 2007). EpiSCs
can be reprogrammed to assume the defining characteristics of ES
cells, including germline competence, by transfecting them with
single pluripotency factors, such as Klf4 (Guo et al., 2009; Hall et
al., 2009). Extended culture in the presence of feeders and LIF can
also reprogramme EpiSCs at low efficiency. This may be
attributable to their reversion to an early epiblast state (Bao et al.,
2009) or to their progression to PGCs, which then convert to EG
cells (Hayashi and Surani, 2009b). Interestingly, naïve pluripotent
cells can be derived from single cells isolated from the mouse postimplantation epiblast and explanted into culture on feeders in
medium supplemented with serum and LIF, but this process
requires a culture period of 2-5 weeks (Bao et al., 2009).
How do ES cells arise?
Because ES cells have been produced from explanted ICMs,
historically they have been equated to ICM cells from 3.5 dpc
mouse blastocysts. However, it is also possible to derive ES cells
from blastomeres isolated from mouse embryos at the eight-cell
stage (Eistetter, 1989). During the process of ES cell derivation
from isolated blastomeres, cell division results in the formation of
a ‘mini-blastocyst’ environment, from which ES cells may
subsequently emerge. The derivation of ES cells directly from
individual ICM cells isolated before segregation of the epiblast and
hypoblast has not been reported. In a study involving the
microdissection of peri-implantation mouse embryos at 4.5 dpc,
Brook and Gardner demonstrated conclusively that the origin of ES
cells is the early epiblast, following its overt segregation from the
hypoblast (Brook and Gardner, 1997). They also showed that ES
cells can be derived directly from single epiblast cells isolated from
freshly retrieved 4.5 dpc embryos, but achieved a maximum of
only three ES cell clones per embryo. This led to speculation that
the process of ES cell derivation may capture a subpopulation of
Postimplantation epiblast,
EpiSCs and human ESCs
the epiblast that contains specified germ cell precursors (Gardner
and Brook, 1997; Zwaka and Thomson, 2005). However, by using
the more permissive culture conditions of 2i medium supplemented
with LIF, the number of ES cell clones that can be obtained from
a single embryo can be increased (Nichols et al., 2009b). This
suggests that all epiblast cells may transiently have the capacity to
become ES cells, although the realisation of this potential depends
upon the timing of their isolation and their culture environment.
Recently published studies have provided evidence that ICM
cells undergo significant changes during the outgrowth phase of ES
cell derivation. A single-cell gene expression analysis, performed
using cells from whole-mouse embryos plated in conventional ES
cell culture conditions, has shown that the molecular profile of a
subgroup of cells changes dramatically as they progress from ICM
to ES cell status (Tang et al., 2010). These changes include the
upregulation of genes that are associated with self-renewal, such as
Lin28a, Tcf3 (transcription factor 3) and Nr0b1 (nuclear receptor
subfamily 0, group B, member 1), and the downregulation of genes
associated with developmental differentiation, such as Gata6, Cdx2
and Hoxd8, coincident with specific alterations in the levels of
certain epigenetic regulators, such as an increase in Dnmt3a (DNA
methyltransferase 3A), Hdac5 (histone deacetylase 5) and Cbx1
(chromobox homologue 1). Interestingly, a proportion of genes
involved in the specification of PGCs, such as fragilis (interferon
induced transmembrane protein 3; Ifitm3 – Mouse Genome
Informatics), Blimp1 and Prdm14, are slightly upregulated during
outgrowth and in the resulting ES cells (Tang et al., 2010). This
observation raises the possibility that in whole-explant culture
under serum-containing conditions, it may be possible for induction
of germ cell precursors to occur. These might potentially be
converted to EG cells by further culture, resulting in the generation
of pluripotent cell lines. Thus, we speculate that there may be two
alternative routes to the derivation of naïve pluripotent cell lines
during the derivation process. The first captures the state of naïve
pluripotency as it arises in the newly formed epiblast; the second
depends upon induction of the germ cell lineage during outgrowth
culture (Fig. 1). It would be interesting to perform single-cell gene
DEVELOPMENT
Key
expression analysis on embryos and their derivative ES cell lines
during the process of ES cell derivation in 2i medium. It might be
anticipated that PGC-associated genes, such as fragilis and Blimp1,
would not be detected if ES cells can be derived directly from the
early epiblast.
The role of embryonic diapause in ES cell
derivation
An interesting property of rodents is their potential for embryonic
diapause, which may be significant for the facility of ES cell
derivation. Diapause is a phenomenon that can occur in mice and
rats when embryos are produced in a suckling mother. In diapause,
the embryos advance to the blastocyst stage, hatch from the zona
pelucida and segregate the epiblast and hypoblast, but then remain
in an unimplanted, non-progressive state until oestrogen is restored.
The epiblast retains expression of Oct4 and Nanog, and both X
chromosomes are active in female embryos (Silva et al., 2009). LIF
signalling is specifically required in the epiblast in diapause,
consistent with there being a close relationship between the early
epiblast and ES cells (Nichols et al., 2001). The implementation of
diapause has been shown to improve the efficiency of ES cell
derivation (Brook and Gardner, 1997; Kawase et al., 1994). This
may be because development is paused when the epiblast is in the
state of naïve pluripotency, thus increasing the likelihood of
capturing this state in culture. From mammals that do not have
diapause, true ES cells have not yet been derived, and little is
known about the details of epiblast development (such as the
relative timing of commitment, the expression of markers and the
reactivation/inactivation status of the X chromosome) that would
facilitate culture optimisation. Does this failure to produce true ES
cells reflect an evolutionary divergence of development in which
the state of naïve pluripotency is rapidly transited or bypassed in
non-murine mammals, or are the current culture conditions
unsuitable to capture this state in vitro? The means to harness naïve
pluripotent cells from non-rodent mammals may require the
development of culture conditions that will promote the
progression of an explanted epiblast cell through development to
PGC specification, and from PGC specification to reprogramming
to an EG cell.
Pluripotent cell lines from human embryos
The overlap in properties between human ‘ES’ cells and EpiSCs
suggests that during the derivation process in humans, the epiblast
may advance to the equivalent of the post-implantation state.
Although these pluripotent human cell lines are widely used for
academic and pharmaceutical research, they tend to be somewhat
restricted in their differentiation capacity and are generally more
difficult to manipulate than are mouse ES cells. Will it be possible
to derive naïve pluripotent human ES cells that more closely
resemble mouse ES cells than mouse EpiSCs? Optimism for the
attainment of this goal is provided by a recent study in which the
key pluripotency factors Oct4, Klf4 and Klf2 were ectopically
induced in human ‘ES’ cells in 2i medium supplemented with LIF
and forskolin. These conditions led to the conversion of these cells
into a cell type that possesses the characteristics of mouse ES cells,
including the possession of two active X chromosomes and a
responsiveness to LIF signalling (Hanna et al., 2010). It remains to
be determined whether ES cells from human embryos could be
captured by devising a strategy to maintain the early epiblast cells
in a state of naïve pluripotency. This may require the isolation of
epiblast cells from trophectoderm and extra-embryonic endoderm
or the deployment of additional kinase inhibitors to prevent
HYPOTHESIS
7
explanted cells from progressing to the state of the postimplantation epiblast. Alternatively, if such attempts are unfruitful,
it may be worth further investigating the possibility of capturing
the PGC-EG transition by inductive explant culture.
Conclusions
We have presented here the possibility that the state of naïve
pluripotency of mouse ES cells may be reached in vitro by two
main alternative routes (captured directly from the nascent epiblast
or via induction of PGCs), which depend upon the culture
conditions used and the cellular environment. This reflects the
complex intertwining of pluripotency and the germline in
mammalian embryos. We propose experimental approaches to
validate this hypothesis that include single-cell molecular profiling
and time-lapse imaging of reporter expression in explanted
embryos or ICMs during ES cell derivation, using alternative
culture regimes and at various time points. The key information to
be gained from these experiments will be whether or not genes
associated with germ cell specification, such as fragilis and Blimp1,
are turned on during ES cell derivation in 2i medium, as was
previously reported for ES cell derivation using feeders and serum
(Tang et al., 2010). If not, it may be concluded that the progression
from ICM to ES cell need not be achieved via PGCs. However, that
these PGC-associated genes have been detected during ES cell
derivation on feeders with serum implies an alternative, inductive
route to attaining naïve pluripotency. To obtain naïve pluripotent
cells from non-murine mammals, monitoring the expression of
these genes may provide a means to optimise protocols for ES cell
derivation that maximise the induction of PGCs in explant cultures.
It may then be possible to develop protocols that convert PGCs to
EG cells. The recent report describing the efficient production of
EG cells from rat embryos using 2i medium may shed light on how
to achieve this goal (Leitch et al., 2010).
Acknowledgements
We thank all members, past and present, of the Smith and Nichols
laboratories, especially Graziano Martello for critical discussion of the
manuscript and Deborah Goode for help with the figures. A.S. is funded by
the Medical Research Council; J.N. is funded by The University of Cambridge,
UK.
Competing interests statement
The authors declare no competing financial interests.
References
Bao, S., Tang, F., Li, X., Hayashi, K., Gillich, A., Lao, K. and Surani, M. A.
(2009). Epigenetic reversion of post-implantation epiblast to pluripotent
embryonic stem cells. Nature 461, 1292-1295.
Batlle-Morera, L., Smith, A. and Nichols, J. (2008). Parameters influencing
derivation of embryonic stem cells from murine embryos. Genesis 46, 758-767.
Beck, F., Erler, T., Russell, A. and James, R. (1995). Expression of Cdx-2 in the
mouse embryo and placenta: possible role in patterning of the extra-embryonic
membranes. Dev. Dyn. 204, 219-227.
Bradley, A., Evans, M. J., Kaufman, M. H. and Robertson, E. (1984). Formation
of germ-line chimaeras from embryo-derived teratocarcinoma cell lines. Nature
309, 255-256.
Brinster, R. L. (1974). The effect of cells transferred into the mouse blastocyst on
subsequent development. J. Exp. Med. 140, 1049-1056.
Brons, I. G., Smithers, L. E., Trotter, M. W., Rugg-Gunn, P., Sun, B., Chuva de
Sousa Lopes, S. M., Howlett, S. K., Clarkson, A., Ahrlund-Richter, L.,
Pedersen, R. A. et al. (2007). Derivation of pluripotent epiblast stem cells from
mammalian embryos. Nature 448, 191-195.
Brook, F. A. and Gardner, R. L. (1997). The origin and efficient derivation of
embryonic stem cells in the mouse. Proc. Natl. Acad. Sci. USA 94, 5709-5712.
Buehr, M. and Smith, A. (2003). Genesis of embryonic stem cells. Philos. Trans. R.
Soc. Lond. B. Biol. Sci 358, 1397-1402.
Buehr, M., Nichols, J., Stenhouse, F., Mountford, P., Greenhalgh, C. J.,
Kantachuvesiri, S., Brooker, G., Mullins, J. and Smith, A. G. (2003). Rapid
loss of Oct-4 and pluripotency in cultured rodent blastocysts and derivative cell
lines. Biol. Reprod. 68, 222-229.
DEVELOPMENT
Development 138 (1)
HYPOTHESIS
Burdon, T., Stracey, C., Chambers, I., Nichols, J. and Smith, A. (1999).
Suppression of SHP-2 and ERK signalling promotes self-renewal of mouse
embryonic stem cells. Dev. Biol. 210, 30-43.
Chambers, I., Colby, D., Robertson, M., Nichols, J., Lee, S., Tweedie, S. and
Smith, A. (2003). Functional expression cloning of Nanog, a pluripotency
sustaining factor in embryonic stem cells. Cell 113, 643-655.
Chazaud, C., Yamanaka, Y., Pawson, T. and Rossant, J. (2006). Early lineage
segregation between epiblast and primitive endoderm in mouse blastocysts
through the Grb2-MAPK pathway. Dev. Cell 10, 615-624.
Cheng, A. M., Saxton, T. M., Sakai, R., Kulkarni, S., Mbamalu, G., Vogel, W.,
Tortorice, C. G., Cardiff, R. D., Cross, J. C., Muller, W. J. et al. (1998).
Mammalian Grb2 regulates multiple steps in embryonic development and
malignant transformation. Cell 95, 793-803.
Damjanov, I., Solter, D., Belicza, M. and Skreb, N. (1971). Teratomas obtained
through extrauterine growth of seven-day mouse embryos. J. Natl. Cancer Inst.
46, 471-475.
Diwan, S. B. and Stevens, L. C. (1976). Development of teratomas from
ectoderm of mouse egg cylinders. J. Natl. Cancer Inst. 57, 937-942.
Durcova-Hills, G., Adams, I. R., Barton, S. C., Surani, M. A. and McLaren, A.
(2006). The role of exogenous fibroblast growth factor-2 on the reprogramming
of primordial germ cells into pluripotent stem cells. Stem Cells 24, 1441-1449.
Eistetter, H. R. (1989). Pluripotent embryonal cell lines can be established from
disaggregated mouse morulae. Dev. Growth Differ. 31, 275-282.
Evans, M. J. and Kaufman, M. (1981). Establishment in culture of pluripotential
cells from mouse embryos. Nature 292, 154-156.
Gardner, R. L. and Brook, F. A. (1997). Reflections on the biology of embryonic
stem cells. Int. J. Dev. Biol. 41, 235-243.
Guo, G., Yang, J., Nichols, J., Hall, J. S., Eyres, I., Mansfield, W. and Smith, A.
(2009). Klf4 reverts developmentally programmed restriction of ground state
pluripotency. Development 136, 1063-1069.
Hall, J., Guo, G., Wray, J., Eyres, I., Nichols, J., Grotewold, L., Morfopoulou,
S., Humphreys, P., Mansfield, W., Walker, R. et al. (2009). Oct4 and LIF/Stat3
additively induce Kruppel factors to sustain embryonic stem cell self-renewal.
Cell Stem Cell 5, 597-609.
Hanna, J., Markoulaki, S., Mitalipova, M., Cheng, A. W., Cassady, J. P.,
Staerk, J., Carey, B. W., Lengner, C. J., Foreman, R., Love, J. et al. (2009).
Metastable pluripotent states in NOD-mouse-derived ESCs. Cell Stem Cell 4,
513-524.
Hanna, J., Cheng, A. W., Saha, K., Kim, J., Lengner, C. J., Soldner, F., Cassady,
J. P., Muffat, J., Carey, B. W. and Jaenisch, R. (2010). Human embryonic stem
cells with biological and epigenetic characteristics similar to those of mouse
ESCs. Proc. Natl. Acad. Sci. USA 107, 9222-9227.
Hayashi, K. and Surani, M. A. (2009a). Resetting the epigenome beyond
pluripotency in the germline. Cell Stem Cell 4, 493-498.
Hayashi, K. and Surani, M. A. (2009b). Self-renewing epiblast stem cells exhibit
continual delineation of germ cells with epigenetic reprogramming in vitro.
Development 136, 3549-3556.
Kawase, E., Suemori, H., Takahashi, N., Okazaki, K., Hashimoto, K. and
Nakatsuji, N. (1994). Strain difference in establishment of mouse embryonic
stem (ES) cell lines. Int. J. Dev. Biol. 38, 385-390.
Kiyonari, H., Kaneko, M., Abe, S. and Aizawa, S. (2010). Three inhibitors of
FGF receptor, ERK, and GSK3 establishes germline-competent embryonic stem
cells of C57BL/6N mouse strain with high efficiency and stability. Genesis 48,
317-327.
Kunath, T., Saba-El-Leil, M. K., Almousailleakh, M., Wray, J., Meloche, S. and
Smith, A. (2007). FGF stimulation of the Erk1/2 signalling cascade triggers
transition of pluripotent embryonic stem cells from self-renewal to lineage
commitment. Development 134, 2895-2902.
Labosky, P. A., Barlow, D. P. and Hogan, B. L. (1994). Mouse embryonic germ
(EG) cell lines: transmission through the germline and differences in the
methylation imprint of insulin-like growth factor 2 receptor (Igf2r) gene
compared with embryonic stem (ES) cell lines. Development 120, 3197-3204.
Leitch, H. G., Blair, K., Mansfield, W., Ayetey, H., Humphreys, P., Nichols, J.,
Surani, M. A. and Smith, A. (2010). Embryonic germ cells from mice and rats
exhibit properties consistent with a generic pluripotent ground state.
Development 137, 2279-2287.
Mak, W., Nesterova, T. B., de Napoles, M., Appanah, R., Yamanaka, S., Otte,
A. P. and Brockdorff, N. (2004). Reactivation of the paternal X chromosome in
early mouse embryos. Science 303, 666-669.
Martin, G. R. (1981). Isolation of a pluripotent cell line from early mouse embryos
cultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl. Acad.
Sci. USA 78, 7634-7638.
Matsui, Y., Zsebo, K. and Hogan, B. L. M. (1992). Derivation of pluripotential
embryonic stem cells from murine primordial germ cells in culture. Cell 70, 841847.
Nichols, J. and Smith, A. (2009). Naive and primed pluripotent states. Cell Stem
Cell 4, 487-492.
Development 138 (1)
Nichols, J., Davidson, D., Taga, T., Yoshida, K., Chambers, I. and Smith, A. G.
(1996). Complementary tissue-specific expression of LIF and LIF-receptor mRNAs
in early mouse embryogenesis. Mech. Dev. 57, 123-131.
Nichols, J., Chambers, I., Taga, T. and Smith, A. (2001). Physiological rationale
for responsiveness of mouse embryonic stem cells to gp130 cytokines.
Development 128, 2333-2339.
Nichols, J., Jones, K., Phillips, J. M., Newland, S. A., Roode, M., Mansfield,
W., Smith, A. and Cooke, A. (2009a). Validated germline-competent
embryonic stem cell lines from nonobese diabetic mice. Nat. Med. 15, 814-818.
Nichols, J., Silva, J., Roode, M. and Smith, A. (2009b). Suppression of Erk
signalling promotes ground state pluripotency in the mouse embryo.
Development 136, 3215-3222.
Okamoto, I., Otte, A. P., Allis, C. D., Reinberg, D. and Heard, E. (2004).
Epigenetic dynamics of imprinted X inactivation during early mouse
development. Science 303, 644-649.
Pelton, T. A., Sharma, S., Schulz, T. C., Rathjen, J. and Rathjen, P. D. (2002).
Transient pluripotent cell populations during primitive ectoderm formation:
correlation of in vivo and in vitro pluripotent cell development. J. Cell Sci. 115,
329-339.
Plusa, B., Piliszek, A., Frankenberg, S., Artus, J. and Hadjantonakis, A. K.
(2008). Distinct sequential cell behaviours direct primitive endoderm formation
in the mouse blastocyst. Development 135, 3081-3091.
Rappolee, D. A., Basilico, C., Patel, Y. and Werb, Z. (1994). Expression and
function of FGF-4 in peri-implantation development in mouse embryos.
Development 120, 2259-2269.
Rastan, S. (1982). Timing of X-chromosome inactivation in postimplantation
mouse embryos. J. Embryol. Exp. Morphol. 71, 11-24.
Rossant, J. (2008). Stem cells and early lineage development. Cell 132, 527-531.
Saitou, M., Barton, S. C. and Surani, M. A. (2002). A molecular programme for
the specification of germ cell fate in mice. Nature 418, 293-300.
Silva, J., Barrandon, O., Nichols, J., Kawaguchi, J., Theunissen, T. W. and
Smith, A. (2008). Promotion of reprogramming to ground state pluripotency by
signal inhibition. PLoS Biol. 6, e253.
Silva, J., Nichols, J., Theunissen, T. W., Guo, G., van Oosten, A. L.,
Barrandon, O., Wray, J., Yamanaka, S., Chambers, I. and Smith, A. (2009).
Nanog is the gateway to the pluripotent ground state. Cell 138, 722-737.
Smith, A. G., Heath, J. K., Donaldson, D. D., Wong, G. G., Moreau, J., Stahl,
M. and Rogers, D. (1988). Inhibition of pluripotential embryonic stem cell
differentiation by purified polypeptides. Nature 336, 688-690.
Solter, D., Skreb, N. and Damjanov, I. (1970). Extrauterine growth of mouse egg
cylinders results in malignant teratoma. Nature 227, 503-504.
Stevens, L. C. (1962). The biology of teratomas including evidence indicating their
origin form primordial germ cells. Annee Biol. 1, 585-610.
Tada, T., Tada, M., Hilton, K., Barton, S. C., Sado, T., Takagi, N. and Surani,
M. A. (1998). Epigenotype switching of imprintable loci in embryonic germ cells.
Dev. Genes Evol. 207, 551-561.
Tang, F., Barbacioru, C., Bao, S., Lee, C., Nordman, E., Wang, X., Lao, K. and
Surani, M. A. (2010). Tracing the derivation of embryonic stem cells from the
inner cell mass by single-cell RNA-Seq analysis. Cell Stem Cell 6, 468-478.
Tesar, P. J., Chenoweth, J. G., Brook, F. A., Davies, T. J., Evans, E. P., Mack, D.
L., Gardner, R. L. and McKay, R. D. (2007). New cell lines from mouse epiblast
share defining features with human embryonic stem cells. Nature 448, 196-199.
Thomas, P. and Beddington, R. (1996). Anterior primitive endoderm may be
responsible for patterning the anterior neural plate in the mouse embryo. Curr.
Biol. 6, 1487-1496.
Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., Waknitz, M. A., Swiergiel,
J. J., Marshall, V. S. and Jones, J. M. (1998). Embryonic stem cell lines derived
from human blastocysts. Science 282, 1145-1147.
Wang, J. K., Gao, G. and Goldfarb, M. (1994). Fibroblast growth factor receptors
have different signaling and mitogenic potentials. Mol. Cell. Biol. 14, 181-188.
Williams, R. L., Hilton, D. J., Pease, S., Willson, T. A., Stewart, C. L., Gearing,
D. P., Wagner, E. F., Metcalf, D., Nicola, N. A. and Gough, N. M. (1988).
Myeloid leukaemia inhibitory factor maintains the developmental potential of
embryonic stem cells. Nature 336, 684-687.
Wray, J., Kalkan, T. and Smith, A. G. (2010). The ground state of pluripotency.
Biochem. Soc. Trans. 38, 1027-1032.
Yamanaka, Y., Lanner, F. and Rossant, J. (2010). FGF signal-dependent
segregation of primitive endoderm and epiblast in the mouse blastocyst.
Development 137, 715-724.
Ying, Q. L., Nichols, J., Chambers, I. and Smith, A. (2003). BMP induction of Id
proteins suppresses differentiation and sustains embryonic stem cell self-renewal
in collaboration with STAT3. Cell 115, 281-292.
Ying, Q. L., Wray, J., Nichols, J., Batlle-Morera, L., Doble, B., Woodgett, J.,
Cohen, P. and Smith, A. (2008). The ground state of embryonic stem cell selfrenewal. Nature 453, 519-523.
Zwaka, T. P. and Thomson, J. A. (2005). A germ cell origin of embryonic stem
cells? Development 132, 227-233.
DEVELOPMENT
8