Extraembryonic Endoderm cells as a model of endoderm development

Review Article

Extraembryonic Endoderm cells as a model of endodermdevelopment

Asja T. Moerkamp,1 Agnieszka Paca,2 Marie-Jos�e Goumans,1 Tilo Kunath,2

Boudewijn P. T. Kruithof1 and Marianna Kruithof-de Julio1,*1Department of Molecular and Cell Biology, Centre of Biomedical Genetics, Leiden University Medical Center, Leiden,

the Netherlands; and 2Medical Research Council Centre for Regenerative Medicine, University of Edinburgh, Edinburgh,UK

In recent years the multipotent extraembryonic endoderm (XEN) stem cells have been the center of much atten-tion. In vivo, XEN cells contribute to the formation of the extraembryonic endoderm, visceral and parietal endo-derm and later on, the yolk sac. Recent data have shown that the distinction between embryonic andextraembryonic endoderm is not as strict as previously thought due to the integration, and not the displace-ment, of the visceral endoderm into the definitive embryonic endoderm. Therefore, cells from the extraembry-onic endoderm also contribute to definitive endoderm. Many research groups focused on unraveling thepotential and ability of XEN cells to both support differentiation and/or differentiate into endoderm-like tissues asan alternative to embryonic stem (ES) cells. Moreover, the conversion of ES to XEN cells, shown recently with-out genetic manipulations, uncovers significant and novel molecular mechanisms involved in extraembryonicendoderm and definitive endoderm development. XEN cell lines provide a unique model for an early mammalianlineage that complements the established ES and trophoblast stem cell lines. Through the study of essentialgenes and signaling requirements for XEN cells in vitro, insights will be gained about the developmentalprogram of the extraembryonic and embryonic endodermal lineage in vivo. This review will provide an overviewon the current literature focusing on XEN cells as a model for primitive endoderm and possibly definitive endo-derm as well as the potential of using these cells for therapeutic applications.

Key words: definitive endoderm, development, extraembryonic endoderm, stem cells, XEN cells.

Introduction: Extraembryonic endodermdevelopment

The mature mouse blastocyst (4.5 days post-coitum

(dpc)) consists of three distinct cell types: the trophec-

toderm, which gives rise to the trophoblast and extra-

embryonic ectoderm (ExEc), the pluripotent cells of the

epiblast, and the primitive or extraembryonic endo-

derm (ExEn), an epithelial layer of cells on the surfaceof the epiblast. The primitive endoderm gives rise to: (i)

visceral endoderm (VE) that surrounds the epiblast and

the ExEc; and (ii) parietal endoderm (PE) that interacts

with the trophoblast giant cell layer. PE cells migratealong the inner surface of the trophectoderm and

together with trophoblast giant cells form the parietal

yolk sac (Hogan et al. 1980). The PE, as well as the

VE, mediates nutrient-waste exchange for the develop-

ing embryo. Initially the VE overlays only the epiblast,

but as the ExEc increases in size the VE quickly

expands to also cover the ExEc. The VE overlaying the

epiblast becomes molecularly and morphologically dis-tinct from the VE in contact with the ExEc around

5.0 dpc, representing the embryonic VE (emVE) and

extraembryonic VE (exVE), respectively. The cells from

the exVE are columnar and cuboidal, while the emVE

cells are flatter and more epithelial in shape (Takito &

Al-Awqati 2004).

Around 5.5 dpc a group of cells at the distal tip of

the epiblast differentiates into a morphologically distin-guishable subset of emVE, the distal visceral endo-

derm (DVE) (Rivera-Perez et al. 2003; Srinivas et al.

2004). This marks the formation of the first axis of the

*Author to whom all correspondence should be addressed.Email: [email protected] 3 December 2012; revised 19 December 2012;

accepted 19 December 2012.ª 2013 The AuthorsDevelopment, Growth & Differentiation ª 2013 Japanese

Society of Developmental Biologists

Develop. Growth Differ. (2013) 55, 301–308 doi: 10.1111/dgd.12036

The Japanese Society of Developmental Biologists

body, the distal–proximal axis. Within 4–5 h (betweenapproximately 5.75 and 6.0 dpc) the DVE migrates

proximally as a continuous epithelial sheet to the pro-

spective anterior pole of the embryo. The underlying

mechanism of this migration is yet to be fully charac-

terized and both active migration and differences in

the proliferation rate of the anterior versus posterior

epiblast have been suggested (Srinivas et al. 2004;

Migeotte et al. 2010; Stuckey et al. 2011; Trichaset al. 2011). The unilateral movement of the DVE

changes the distal–proximal axis into the anterior–posterior axis of the embryo and the DVE is now

called anterior visceral endoderm (AVE).

The VE and its derivatives, play critical roles in orga-

nization and differentiation of the epiblast. The VE is

the first site of hematopoiesis (Toles et al. 1989; McG-

rath & Palis 2005) and induces through the expressionof Indian hedgehog and vascular endothelial growth

factor the formation of blood islands and endothelial

cells (Dyer et al. 2001; Byrd et al. 2002; Damert et al.

2002). In addition, the proximal VE was shown to be

involved in early primordial germ cell differentiation (de

Sousa Lopes et al. 2004, 2007). Finally, microsurgical

removal of AVE resulted in anterior neural structures

truncations (Thomas & Beddington 1996) and itsderived BMP2 signals have been shown to take part in

heart positioning and foregut invagination (Madabhushi

& Lacy 2011).

Interestingly, the lineage distinction between embry-

onic and ExEn tissue was marked by the assumption

that the VE that surrounds the epiblast was displaced

by the definitive endoderm. Recently it has been

shown that cells from the VE persist within the defini-tive endoderm layer of the embryo and contribute to

the early gut tube. This suggests that the distinction

between extraembryonic and embryonic tissues is not

as strict as believed and the lineage that was previ-

ously considered to be exclusively embryonic has

extraembryonic contributions (Kwon et al. 2008). An

interesting question is whether, within the definitive

endoderm and potentially its derivative tissues, thereare molecular and functional differences between the

ExEn- and epiblast-derived cells that may be studied

by in vitro culturing and manipulating cells representa-

tive of the ExEn.

Derivation, properties and applications ofExEn-derived stem cells

Stem cells can be derived from each of the primary lin-

eages of the mammalian embryo (Fig. 1). ES cells from

the inner cell mass (ICM) or early epiblast, trophoblast

stem (TS) cells from the trophectoderm layer and XEN

stem cells from the primitive endoderm (or ExEn). Most

importantly each one of these stem cell systems arecapable of indefinite self-renewal in culture and once

reintroduced into the mouse embryo will display line-

age restricted contributions in the resulting chimeric

embryos that are consistent with their lineage of origin

(Beddington & Robertson 1989; Tanaka et al. 1998;

Kunath et al. 2005). Interestingly, in vivo, XEN cells

can only repopulate the PE, rarely the VE (Kunath

et al. 2005; Kruithof-de Julio et al. 2011).Initially, parietal endoderm cell (PEC) lines were iso-

lated by Fowler et al. (1990). In vitro studies have

shown that these cells have characteristics of PE, clo-

sely resembling the basement membrane matrix of

Reichert’s membrane. However, their chimera contri-

bution potential was not assessed (Fowler et al. 1990).

PECs morphologically resemble the more recently

isolated XEN cells (Kunath et al. 2005; reviewed inRossant 2007) being round and refractile or stellate or

epithelial-like cells. Several methods of isolation have

been proposed for XEN cell derivation, which lead all

to cells with similar morphological characteristics; how-

ever, their response to growth factors seems to be

influenced by the derivation process. In this context, it

is essential to appreciate that primitive endoderm

already exhibits heterogeneous expression of some ofthe DVE/AVE markers, such as Cer1 and Lefty1 (Tak-

aoka et al. 2006, 2011; Torres-Padilla et al. 2007).

Furthermore, Nodal/Activin signaling can be differen-

tially perceived within the primitive endoderm (Granier

et al. 2011). This would imply that, at the time of sig-

naling pathway maturation, primitive endoderm cells

are sensitive to even minor changes in signaling inten-

sity. Interestingly, primitive endoderm-progenitorsexpressing Oct4 exhibit greater developmental plastic-

ity than Oct4-expressing epiblast progenitors at a simi-

lar stage (Grabarek et al. 2012) which could reflect the

in vitro heterogeneous nature of XEN cells.

Extensive microarray analysis on XEN cells per-

formed by several groups all agree in the expression of

primitive endoderm markers SOX7, GATA4, GATA6

and the VE markers hHEX and DKK1. Furthermore,XEN cells are characterized by the lack of AFP expres-

sion as well as the absence of definitive endoderm

markers (Kunath et al. 2005; Brown et al. 2010b; Kru-

ithof-de Julio et al. 2011). Given their reproducible der-

ivation from the ExEn and their expression profile

similar to this extraembryonic tissue, XEN cells can be

a powerful tool to study inductive effects attributed to

the AVE. Brown et al. (2010a,b) have undertaken anextensive array analysis of three cell lines that are simi-

lar to the heart-inducing AVE: two embryonal carci-

noma-derived (END2 and PYS2) and the XEN cells. By

comparing the gene expression profiles they have

identified a discrete set of genes that could support

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Development, Growth & Differentiation ª 2013 Japanese Society of Developmental Biologists

302 A. T. Moerkamp et al.

myocardial differentiation. In addition, by using XEN

cell-derived conditioned media on embryoid bodies

they were able to enhance cardiogenesis (Brown et al.

2010a). These are interesting observations with poten-

tial therapeutic properties. In order to obtain the

appropriate cell products (in this context, cardiomyo-cytes for cardiac repair), it is of importance to stepwise

direct the fate of the stem cell of interest with the

proper instructive signals in vitro. During development,

the VE interacts with the nascent mesoderm to induce

the cardiac fate. Therefore, XEN cells could be used to

recapitulate this developmental process in vitro and

induce and accelerate differentiation of the susceptible

stem cells into the cardiac lineage.Although derived from primitive endoderm, XEN cells

contribute efficiently in chimeras to PE but not to VE

(Kunath et al. 2005; Kruithof-de Julio et al. 2011). This

lack of contribution to the VE lineage could be caused

by many factors, including the preferential interaction

with the mural trophectoderm (Artus et al. 2012; Kruit-

hof-de Julio and Shen, unpubl. data, 2009), the alter-

ation of a signaling pathway in the establishment ofthe cell line based on the derivation process (XEN cells

derived in the presence of LIF poorly respond to

growth factor stimulation) or the derivation of a “com-

mitted” cell that has lost potency. In support of the lat-

ter, extraembryonic endoderm precursor (XEN-P) stem

cells have been derived from rat blastocyst (Debeb

et al. 2009; Galat et al. 2009). These cells are charac-

terized by a less “endoderm” defined gene expression

profile; they express the ES markers OCT4, REX1, AP,

and SSEA1 and contribute to the PE as well as the VE

lineages in chimeras. The authors suggest they are

precursor endoderm cells as they could represent the

first committed step of the ExEn.

Finally, with respect to the plasticity or commitmentof stem cells, recently Cho et al. (2012) was able to

derive XEN cells from mouse ES cells via the addition

of exogenous retinoic acid and Activin. These XEN

cells are indistinguishable from embryo-derived XEN

cells, including their differentiation capacity (Cho et al.

2012). These data show a high degree of plasticity

within the ICM and provide the possibility of deriving

XEN cell lines from the various mutant ES cell linesavailable, thereby shedding light on the factors

required for XEN cell derivation and the development

of ExEn.

Signaling pathways in XEN cells

Characteristic of XEN cells are the transcription factors

GATA4 and GATA6, specifically expressed in theExEn. GATA6 is present from 3.5 dpc in the ICM in a

salt and pepper distribution with NANOG and then

restricted to the primitive endoderm (Chazaud et al.

2006). Gata6-deficient mice are embryonically lethal

and Gata6-null ES cells fail to specify VE in vivo and

in vitro, suggesting a key role for GATA6 in both VE

and PE differentiation (Morrisey et al. 1998). Forced

and maintained expression of the Gata4 and Gata6 in

Fig. 1. At 3.5–4.5 days post-coitum (dpc), trophoblast stem (TS) cells, embryonic stem (ES) cells and extraembryonic endoderm (XEN)/

parietal endoderm cells (PEC) can be derived from the trophectoderm, inner cell mass (ICM) and primitive endoderm, respectively, and

propagate indefinitely in culture. In chimeras, TS, ES and XEN cells contribute to their lineage of origin. The trophectoderm will give rise

to the placenta; the ICM will form the three germ layers of the embryo and the primitive endoderm will develop into the visceral

endoderm and parietal endoderm lineages.

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XEN cells as a model of endoderm development 303

mouse ES cells mimic XEN cell characteristics bothin vivo and in vitro (Fujikura et al. 2002; Shimosato

et al. 2007), implying that GATA factors play a crucial

role in XEN cell specification. Recently, an essential

stemness factor that has been identified in XEN cells is

SALL4. In fact, XEN cells cannot be derived from

Sall4-null mice. Given it lies upstream of GATA4 and

GATA6, SALL4 seems to play a role as an activator of

key lineage-defining genes in the ExEn. This partiallyexplains why loss of Gata4 or Gata6 leads to a VE

defect and not to a broader loss of primitive endoderm

(Lim et al. 2008).

Interestingly, human ES (hES) cells can also mimic

ExEn and definitive endoderm by constitutively

expressing SOX7 or SOX17, respectively (Seguin et al.

2008). In addition, differentiation of these cells by

either BMP4 treatment in SOX7 overexpressing hEScells increased expression of ExEn markers, whereas

Activin A treatment in SOX17 overexpressing hES cells

increased expression of definitive endoderm markers

as expected for hES cells. Both cell lines, however,

maintained expression of both NANOG and OCT4 in

their undifferentiated state. This suggests a similarity of

the SOX7 overexpressing hES to the XEN-P cells

rather than to the mouse XEN cells and implies a rolefor the SOX transcription factors in maintaining a pre-

cursor state in the cells preventing them from differen-

tiation. Sox17 is expressed in mouse-derived XEN

cells and plays a role in their establishment since XEN

cells cannot be derived from Sox17-null embryos.

However, in Sox17 mutant mice no significant effect

on ExEn development was observed, which the

authors attributed to the highly regulative environmentof the mouse embryo and the continuous expression

of Gata6 and Sox7 in Sox17-deficient embryos (Niakan

et al. 2010).

Endogenous non-coding small RNAs (miRNAs) play

a role in the pluripotency regulatory network of ES, TS

and XEN cells (Marson et al. 2008; Xu et al. 2009;

Spruce et al. 2010). Interestingly, Dicer mutants do not

correctly pattern the VE. In addition, depletion ofmiRNAs in XEN cells leads to a loss of multipotency,

which seems to be mediated via the modulation of the

ERK1/2 signaling pathway and is contrary to what

occurs in embryonic-derived cells. This suggests, for

the extraembryonic lineage, a crucial role for miRNAs

in establishment and maintenance of self-renewal

capability (Spruce et al. 2010). Furthermore, extra-

embryonic tissues have been reported to be hypome-thylated when compared to embryonic tissue

(Chapman et al. 1984; Monk et al. 1987; Gardner &

Davies 1992). Similarly, XEN cells have been recently

shown to express low levels of repressive chromatin

modifications, such as H3K27me3 (Rugg-Gunn et al.

2010). Low methylation of ExEn could potentially givethis tissue a significant competence to easily undergo

differentiation or transdifferentiation.

The lack of XEN cell contribution to VE has inter-

ested several groups and lead to the findings that

Nodal and BMP4, both members of the transforming

growth factor (TGF)b superfamily, treatments direct

XEN cells toward a VE phenotype in vivo and in vitro

(Kruithof-de Julio et al. 2011; Artus et al. 2012; Pacaet al. 2012). Nodal binds to type I (ALK4) or type II

receptors in the presence of the co-receptor Cripto or

Cryptic (members of the EGF-CFC family). Its signal is

propagated to the nucleus via phosphorylation of

SMAD2 and is tightly regulated by inhibitors including

Lefty and Cerberus. XEN cells, treated with either

Nodal or Cripto can be differentiated into a VE pheno-

type in vitro, and in chimeras they contribute moreefficiently to VE. This contribution, however, is not VE

exclusive, which could be due to the highly heteroge-

neous population of XEN cells. In XEN cells, Nodal

signals solely via the ALK4 receptor and the EGF-CFC

Cryptic, as both SB431542 (as inhibitor of the ALK4

receptor) treated XEN cells and XEN cells derived from

Cryptic-null mice do not respond to Nodal. Cripto is,

at least partially, Nodal-independent as its functioncannot be inhibited by SB431542. The authors pro-

pose two possible mechanistic scenarios: an alterna-

tive low affinity receptor or a non-canonical pathway

through an unknown signal transducer (Kruithof-de

Julio et al. 2011). Interestingly, similar conclusions

have been independently reached by Clements et al.

(2011). The authors focus on the cross talk between

the Nodal/Activin signaling pathway and the MAPKp38 in XEN cells revealing a novel role for p38 in regu-

lating Nodal thresholds. They observed that the activa-

tion of p38 is not ALK4, 5 or 7 dependent as it cannot

be inhibited by SB431542; however, it can be trig-

gered by Cripto. This suggests that the non-canonical

pathway by which Cripto functions may be p38

dependent (Clements et al. 2011).

As previously mentioned, BMP4 treatment alsodirects XEN cells to a VE phenotype. In this case

through the canonical BMP receptors, as the chemical

inhibitor Dorsomorphin prevented the VE induction

mediated by BMP4. Interestingly, the mainly induced

VE subtype resembles the VE adjacent to ExEc, and

not emVE (Artus et al. 2012; Paca et al. 2012). This

observation fits with the known expression of BMP4 in

the early embryo, which is highest in the ExEc closestto the proximal epiblast (Lawson et al. 1999). BMP4

was also capable of inducing VE differentiation of PE,

showing that this cell type is not terminally differenti-

ated, and retains the ability to form VE (Artus et al.

2012; Paca et al. 2012).

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Conclusions

Multipotent stem cells have the potential to develop

into different cell types in the body during early life and

growth. They are distinguished from other cell types bytwo important characteristics. First, they can self-

renew and second, under certain physiologic or exper-

imental conditions, they can be induced to become

tissue- or organ-specific cells with special functions.

In order to fulfill the desire to generate a cell-based

therapy, it is necessary to manipulate the cells, in vitro,

to successfully differentiate them towards the cell-type

of interest. The meticulous application of developmen-tal principals to stem cell culture systems is the basis

of this kind of research. The major limitation is the very

low frequency of differentiated cells identified and the

cellular heterogeneity. Only when large numbers of

highly enriched progenitors are accessible, methods

can be defined for their maturation and their functional

capacity rigorously tested in animal models. XEN cells

might give the missing link in this differentiation pro-cess.

Extraembryonic and embryonic tissues interact tospecify each other’s commitment. By using XEN cells

as an inductive feeder layer for ES cells, epiblast

stem cells (EpiSC) or their multipotent progeny, a par-

ticular developmental or differentiation fate can be

induced (Kruithof-de Julio, Moerkamp and Goumans,

unpubl. data, 2012). In this context, XEN cells may

not only be a model to understand ExEn develop-

ment, but also to elucidate the inductive role of theExEn in embryonic tissue specification, for example,

the formation of heart and blood lineages (Brown

et al. 2010a; Artus et al. 2012). Once established,

their roles in these inductive processes, mutant lines

can be derived to pin point the specific genes

involved. Thereby, XEN cells may be a powerful

in vitro tool (Fig. 2), by either the use of conditioned

medium or co-culture, for providing an overview ofthe factors that support the differentiation during

developmental processes, like cardiogenesis and

hematopoiesis. The identification of the exogenous

differentiation stimuli may be translated into clinical

practice for stem cell-based approaches to efficiently

Fig. 2. Extraembryonic endoderm (XEN) cells, or its derived conditioned medium (CDM) may be a valuable in vitro tool to study develop-

mental processes including the communication between the visceral endoderm (VE) and mesoderm as well as these cells may have

therapeutic properties. Understanding the inductive factors underlying mesoderm specification, like cardiogenesis and hematopoiesis,

may be useful for stem cell-based therapeutic approaches.

ª 2013 The Authors



obtain a high number of the differentiated cell type ofinterest. For cell-based therapies, XEN cells are pref-

erably derived from human embryos. However, the

derivation from human embryos was only partly

supplemented by the SOX7 overexpressing hES cells

and remains subject to future studies. Alternatively,

for the identification of developmental stimuli, XEN

cells could be derived from the mouse postimplanta-

tion epiblast. Mouse EpiSC are highly similar to hEScells in their pluripotent state and growth factor

requirements and therefore its derived XEN cells may

resemble human ExEn development more closely.

Cho et al. (2012) was unable to derive XEN cells from

mouse EpiSC by using the same protocol as for the

derivation of XEN cells from mouse ES cells (Cho

et al. 2012). In the case of EpiSC, due to their epige-

netic state, XEN cell derivation may require differentexogenous stimuli. However, there remains the possi-

bility that EpiSC are indeed unable to convert their

developmental fate. Finally, although it has not been

shown to date, XEN cells may be derived from

somatic cells via direct reprogramming, thereby

circumventing the requirement of human embryos.

Although one must keep in mind the highly hetero-

geneous population and the existence of species-spe-cific differences, which may be due to the stage and

derivation protocols, XEN cells can also be a model to

study extraembryonic contribution to definitive endo-

derm and its differentiated derivatives. This raises the

question as to whether XEN cells per se can indeed

contribute to the primitive gut tube or must they be

prior differentiated towards a definitive endodermal

lineage. In later stage chimeras derived from Nodal,Cripto or untreated XEN cells contribution to definitive

endoderm has never been observed (Kruithof-de Julio

and Shen, unpubl. data, 2010), only the contribution

to the visceral yolk sac.

In summary, XEN cells provide a valuable cell culture

model to dissect the extraembryonic endodermal line-

age and its potential contribution to the embryonic

one. Thereby, XEN cells may increase our understand-ing of the molecular mechanism behind endoderm

behavior and function during development. Further-

more, XEN cells may give an insight into the inductive

factors underlying mesoderm specification, like cardio-

genesis and hematopoiesis, which may be reflected

into stem cell-based therapeutic approaches.

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Extraembryonic Endoderm cells as a model of endoderm development