1 Laboratory for Stem Cell Biology, RIKEN Center for Developmental Biology,
2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, 650-0047, Japan
2 Department of Gastroenterology and Hepatology, Graduate school of Medicine,
Kyoto University, 54 Shogoinkawara-cho, Sakyo-ku, Kyoto, 606-8507, Japan
3 Department of Bioinformatic Engineering, Graduate School of Information
Science and Technology, Osaka University, 2-1 Yamada-oka, Suita City, Osaka,
565-0871, Japan
4 Laboratory for Animal Resources and Genetic Engineering, RIKEN Center for
Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, 650-0047,
Japan
* Author for correspondence (e-mail: tera{at}cdb.riken.jp)
Accepted 19 July 2005
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SUMMARY |
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Key words: ES cell, Mesendoderm, Goosecoid, Endoderm, Mouse
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Introduction |
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During early mouse development, endoderm precursors initially arise from
the anterior primitive streak, which corresponds to early and mid gastrula
organizer (EGO and MGO) (Kinder et al.,
2001; Lawson et al.,
1991
; Wells and Melton,
1999
). Fate analyses by the transplantation of these organizers
(Kinder et al., 2001
) and fate
maps defined by cell labelling experiments
(Lawson et al., 1991
) revealed
that cells in two organizers subsequently gave rise to anterior definitive
endoderm and axial mesoderm, including prechordal and notochordal plates.
Although the results from tissue fate analyses strongly suggested that the
organizer contains both endoderm and mesoderm precursors, the presence and the
differentiation course of bi-potent mesendoderm are still elusive because of
the difficulty in isolating a sufficient number of cells for further analyses.
As mesendoderm is implicated as the major source of anterior endoderm in
mammalian development, a part of which can eventually differentiate into
hepatocytes and pancreatic ß-cells
(Wells and Melton, 1999
), its
characterization is essential for elucidating the differentiation pathway
leading to anterior definitive endoderm and also for developing methods to
induce endodermal cells for regenerative medicine.
In mouse development, endoderms are divided into two types: one is visceral
endoderm, which diverges directly from the inner cell mass and gives rise to
extra-embryonic endoderm; and the other is definitive endoderm
(Lu et al., 2001). It is still
difficult to judge whether endodermal cells generated in ES cell cultures
represent definitive or visceral endoderm, owing to lack of molecular markers
distinguishing two endodermal lineages
(Grapin-Botton and Melton,
2000
; Tam et al.,
2003
). This deficit of molecular markers may be circumvented if
the history of the resulting endodermal cells can be defined, as anterior
definitive endoderm is known to be derived from endoderm precursors present in
EGO and MGO (Kinder et al.,
2001
). Recently, Kubo and his colleagues used brachyury (T) as a
marker for primitive streak mesoderm and showed that T-GFP-expressing cells in
ES cell cultures could give rise to cells expressing endodermal markers
(Kubo et al., 2004
). Given
that visceral endoderm is not derived from T+ primitive streak,
this is the first unequivocal demonstration that definitive endoderm cells can
be generated in ES cell cultures. As T is one of early markers for mesoderm,
this suggests that endoderm cells are derived from mesoderm expressing T. In
addition, because T is expressed in both node and early mesodermal lineages
(Showell et al., 2004
), cells
marked by T cannot distinguish mesendoderm from other mesoderm cells. Thus, it
is still necessary to establish new markers that enable us to specifically
track the differentiation course of mesendoderm in an in vitro ES cell
culture.
The goosecoid gene (Gsc) is an ideal marker for mesendoderm, as it
is expressed specifically in the organizer region from which definitive
endoderm arises (Blum et al.,
1992). In addition, Gsc-null mice show no obvious
abnormality during gastrulation (Yamada et
al., 1995
), indicating that insertion of a marker gene into a
Gsc allele may not affect the differentiation process.
The central aim of this study is to identify and characterize bi-potent
mesendoderm and its differentiation process in in vitro ES cell
differentiation system. To achieve this, we have established ES cell lines
that contain a Gsc allele to which an enhanced green fluorescence
protein (gfp) gene (Zhang et al.,
1996) is knocked-in by homologous recombination. We demonstrate
that a defined culture condition allows us to induce almost pure
Gsc+ population. This study shows that bi-potent mesendoderm cells
do exist in our ES cell differentiation culture and are defined as a
Gsc+E-cadherin(ECD)+PDGFR
(
R)+
population that subsequently diverges to
Gsc+ECD+
R- and
Gsc+ECD-
R+ intermediates that
eventually differentiate into definitive endoderm and mesodermal lineages,
respectively.
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Materials and methods |
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Maintenance of ES and OP9 cells
EB5 (a kind gift from Dr Hitoshi Niwa, RIKEN, Kobe, Japan) is a subline
derived from E14tg2a ES cells. This line was generated by targeted integration
of an Oct3/4-IRES-BSD-pA vector into the Oct3/4 allele and carries
the blasticidin S-resistant selection marker gene driven by the
Oct3/4 promoter, which is active under the undifferentiated status
(Niwa et al., 2000).
Undifferentiated ES cells were maintained on gelatin-coated dishes in Glasgow
minimum essential medium (G-MEM; GIBCO-BRL) supplemented with 1% foetal calf
serum (FCS), 10% knockout serum replacement (KSR; GIBCO-BRL), 0.1 mM
nonessential amino acids, 1 mM sodium pyruvate (GIBCO-BRL), 0.1 mM
2-mercaptoethanol (2ME), 1000 U/ml leukemia inhibitory factor (LIF)
(GIBCO-BRL) and 20 µg/ml blasticidin S to eliminate differentiated cells.
OP9 stromal cells were maintained in
minimum essential medium (MEM)
(GIBCO-BRL) supplemented with 20% FCS (Era
and Witte, 2000
).
In vitro ES cell differentiation
Under serum-containing condition, induction of ES cell differentiation was
carried out as described previously
(Nishikawa et al., 1998).
Briefly, undifferentiated ES cells were transferred to type IV collagen-coated
dishes (BioCoat; Becton Dickinson Labware) or non-coated dishes and incubated
in
MEM supplemented with 10% FCS and 50 µM 2ME in the absence of
LIF.
For the induction under serum-free condition, ES cells were seeded onto
type IV collagen-coated 10 cm dishes at a density of 1x105
cells per dish in SF-O3 medium (SFM) (Sanko Junyaku)
(Takakura et al., 1996a)
supplemented with 0.1% bovine serum albumin (BSA) and 50 µM 2ME. For the
formation of EB, ES cells were seeded on non-coated 6 cm petri dishes at a
density of 3x104 cells per dish. In some experiments,
recombinant human activin A, BMP4 and recombinant mouse Nodal (R&D
Systems) were added from the beginning of the culture: activin A 10 ng/ml,
Bmp4 10 ng/ml and nodal 1000 ng/ml.
For re-culture experiments, Gsc+ECD+ and
Gsc+ECDlow cells in Gscgfp/+ES cell
culture or ECD+R+ cells in unmanipulated EB5 ES
cell culture were sorted at day 4 and re-cultured in SFM with activin A on
type IV collagen-coated dishes. For the induction to albumin-producing cells,
day 6 Gsc+ECD+ cells were sorted and re-cultured for 3
days on type I collagen-coated dishes (BioCoat; BD Labware) in SFM with 20
ng/ml Egf (R&D Systems), 20 ng/ml Bmp4, 10 ng/ml acid Fgf (R&D
Systems) and 5 ng/ml basic Fgf (R&D Systems)
(Jung et al., 1999
;
Zaret, 2002
). The
Gsc+ECD+-derived cells were stained by anti-albumin
antibody. The expression of endoderm, hepatic and pancreatic markers was
analyzed by RT-PCR.
Gsc+ECD- cells were sorted at day 6 and used for the
analyses of gene expression and cell fate. For osteogenesis, sorted cells were
re-cultured on gelatin-coated dish in KnockOut D-MEM (GIBCO) supplemented with
10% FCS, 0.1 µM dexamethasone, 50 µM ascorbic-acid-2-phosphate (Sigma),
10 mM ß-glycerophosphate (Sigma) and 10 ng/ml Bmp4
(Pittenger et al., 1999). On
day 28, Gsc+ECD--derived cells were harvested and
stained by Alizarin Red (Muraglia et al.,
2003
). In addition, total RNA was isolated to examine the
expression of bone-specific markers. For vasculogenesis, 500
Gsc+ECD- cells were re-cultured onto a confluent OP9
cell layer in each well of 24-well plate with
MEM containing 10% FCS
and 50 µM 2ME (Hirai et al.,
2003
). Two days later, the number of endothelial colonies was
enumerated after staining wells with either anti-VE-cadherin or anti-Pecam1
antibodies.
Establishment of endoderm cell lines
Gsc+ECD+ cells were sorted at day 6 and re-cultured
in MEM supplemented with 10% FCS and 50 µM 2ME on type I
collagen-coated dishes. The passage of cells was performed every 3 days.
Cell staining and cell sorting
Preparation of the rat monoclonal Abs APA5 (anti-PDGFR)
(Takakura et al., 1996b
),
ECCD2 (anti-ECD) (Shirayoshi et al.,
1986
), AVAS12 (anti-Vegfr2)
(Kataoka et al., 1997
) and
APB5 (anti-PDGFRß) (Sano et al.,
2001
), and their labelling by either biotin or allophycocyanin
were carried out as described previously
(Nishikawa et al., 1998
).
Cultured cells were harvested with cell dissociation buffer (GIBCO-BRL). Cell
staining, analysis by FACS Calibur and cell sorting by FACS Vantage or FACS
Aria (Becton Dickinson) were as previously described
(Nishikawa et al., 1998
).
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Immunostaining of cultured cells
For immunohistochemistry, cultured cells were fixed with 2%
paraformaldehyde in PBS. The following antibodies were used: ECCD2, 1:1000;
AVAS12, 1:1000; APB5, 1:1000; anti-HNF3ß (Foxa2) (goat polyclonal) (Santa
Cruz), 1:100; anti-Gata4 (goat polyclonal) (Santa Cruz), 1:500; anti-brachyury
(goat polyclonal) (Santa Cruz), 1:250; anti-Pecam1 (rat monoclonal) (BD
Pharmingen), 1:500; VECD1 (anti-VE-Cadherin) (rat monoclonal)
(Matsuyoshi et al., 1997),
1:500; anti-albumin (goat polyclonal) (Bethyl), 1:500; anti-cytokeratin 18
(mouse monoclonal) (Progen), 1:200; anti-GFP (rabbit polyclonal) (Molecular
Probes), 1:500. Appropriate HRP-conjugated secondary antibodies were selected,
and the signals were detected by DAB-Ni
(Takakura et al., 1996b
). For
immunofluorescence staining, samples were stained by appropriate
fluorescence-tagged secondary antibodies and examined on Radiance2100 (BioRad)
confocal imaging system.
Tetraploid embryonic complemention
Tetraploid embryo complementation was performed as described
(Nagy et al., 1993;
Tam and Rossant, 2003
), using
C57BL/6 zygotes. Briefly, two-cell stage embryos were electrofused and
developed in vitro to 4N blastocyst stage. Gscgfp/+ ES
cells were injected into blastocysts and transferred to pseudopregnant ICR
females. The expression of GFP was examined in mid-streak stage embryos.
Single cell deposition assay for differentiation potential of mesendodermal cells
Single Gsc+ECD+ cell in day 4 culture was seeded into
individual wells of 96-well collagen IV-coated plates (Becton Dickinson) by
FACS Vantage or Aria equipped with single cell deposition device. Sorted
single cell was cultured in serum-containing medium supplemented with 50 ng/ml
Activin A, 5 ng/ml basic Fgf and 1 mM LiCl for 3 days. Cells were stained by
an antibody cocktail of anti-Foxa2 Ab, APB5 and AVAS12. HRP-conjugated
anti-goat IgG (Sigma) and ALP-conjugated anti-rat IgG (Jackson ImmunoResearch)
were used as secondary antibodies. The substrate used was DAB-Ni in HRP
staining. ALP staining was performed by the Vector Red substrate kit I
(VECTOR), according to manufacturer's protocol.
Transplantation to immunodeficient mice
Endoderm cell line was maintained in MEM media supplemented 10% FBS
and 2ME. Cells of this cell line (1x106) were injected into
either the renal capsule or the spleen of scid/scid immunodeficient mice.
Every week from 2 to 5 weeks after transplantation, mice were sacrificed and
the serial sections of transplanted tissues were investigated.
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Results |
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In order to examine whether or not Gscgfp/+ ES cell
lines can be used for monitoring the differentiation of Gsc+ cells,
we cultured Gscgfp/+ ES cells under conditions that have
been used for inducing mesoderm cells from ES cells
(Nishikawa et al., 1998).
GFP+ cells were detected after 4 days of differentiation in
serum-containing medium (SCM) either on collagen IV-coated dishes or using
embryoid body (EB) formation (Burkert et
al., 1991
) (Fig.
1B, part i). Cell-sorting and RT-PCR analysis of GFP+
and GFP- cells demonstrated that GFP expression correlated with
that of endogenous Gsc. Moreover, expression of other molecular
markers of EGO and MGO, such as Foxa2
(Monaghan et al., 1993
),
Lim1 (Shawlot and Behringer,
1995
) and chordin (Sasai et
al., 1994
), was detected only in GFP+ population, and
expression of T in this population suggests the possibility that it
is not derived from visceral endoderm
(Showell et al., 2004
;
Tam et al., 2003
;
Wilkinson et al., 1990
)
(Fig. 1B, part ii). To check
GFP expression in vivo, we injected Gscgfp/+ ES cells into
blastocysts of tetraploid embryos (Nagy et
al., 1993
; Tam and Rossant,
2003
). In the mid-streak stage embryo, GFP expression was
detectable not only in the gastrula organizer, but also in the newly formed
mesoderm (Fig. 1B, part iii).
The expression pattern is consistent with a previous study of
Gsclacz expression in mouse embryo
(Kinder et al., 2001
). This
result, together with gene expression pattern of Gsc+ cells in
culture, indicates strongly that Gsc+ cells correspond to the
organizer region of actual embryos.
Selective induction of Gsc+ cells in a serum-free medium containing activin
Under the culture condition that we have been using for inducing mesoderm
cells (Nishikawa et al.,
1998), the proportion of GFP+ cells did not exceed 3%
on day 4 (Fig. 1B, part i).
As activin was shown to induce the expression of Gsc in Xenopus
animal cap cells (Cho et al.,
1991; Symes et al.,
1994
), we investigated the effect of activin. The differentiation
of ES cells were induced on collagen IV-coated dishes in SF-O3 serum-free
medium (SFM) with 10 ng/ml of activin. Activin acts as a potent inducer of Gsc
under serum-free culture condition, with 60% and nearly 100% of cells being
GFP+ by day 4 and day 6, respectively
(Fig. 1C, part i). The
expression analysis by RT-PCR showed that a set of molecules specific for the
organizer, including Gsc, Foxa2, Lim1 and chordin, was exclusively
expressed in GFP+ population and in the same population under
serum-containing conditions (Fig.
1C, part ii). Though our ES cell line can differentiate into
neuroectodermal cells by treatment with retinoic acid
(Ying et al., 2003
), the
expression of neuroectodermal markers such as Pax6
(Hill et al., 1992
) and
Sox1 (Wood and Episkopou,
1999
) was not induced in this culture system (data not shown).
Next, we examined whether or not visceral endoderm marker expression was
induced in this serum-free culture, as Gsc is known to be expressed also in
some visceral endoderm cells (Perea-Gomez
et al., 1999
). The expression of visceral endoderm markers such as
Sox7 (Kanai-Azuma et al.,
2002
), Hnf4 (Duncan et
al., 1994
) and Pthr1
(Verheijen et al., 1999
) was
not induced in serum-free culture with activin
(Fig. 1C, part iii). Moreover,
we recently established a culture condition that permits only differentiation
into visceral endoderm (M. Yasunaga and S.T., unpublished). Although Sox7,
Hnf4, Pthr1, Pem (Tam et al.,
2004
) and Msg1
(Dunwoodie et al., 1998
) were
expressed in cells induced under this selective condition for visceral
endoderm, these markers were not in Gsc+ population induced under
activin containing serum-free condition (M. Yasunaga and S.T., unpublished).
These results, together with the result that T was expressed in both
GFP+ and GFP- population
(Fig. 1C, part iv), strongly
suggest that GFP+ population produced under this serum-free
condition does not contain visceral endoderm.
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Gsc+ cells were induced from ES cells, and surface expression of
ECD and R was analyzed at various time points. Initially, most nascent
GFP+ cells co-expressed ECD and
R, but diverged quickly to
ECD+
R- and ECD-
R+
populations (Fig. 2A,B).
ECD-
R+ cells were expressing other mesodermal
lineage markers such as PDGFRß (ßR)
(Betsholtz et al., 2001
) and
Vegfr2 (Kataoka et al., 1997
)
(Fig. 2C).
To confirm the sequence of events occurring during the differentiation of
Gsc+ECD+ cells, we purified
GFP+ECDhigh, GFP+ECDlow and
GFP- cells from day 4 cultures and re-cultured them. In contrast to
the Gsc+ECDhigh population that could give rise to both
Gsc+ECD+ and Gsc+ECD- cells, the
Gsc+ECDlow fraction could generate only
ECDlow/- cells. (Fig.
2D). The Gsc- population contained immature cells that
could differentiate into both Gsc+ECD+ and
Gsc+ECD- populations, but such immature cells
disappeared by day 5 of the culture (data not shown). This re-culture
experiment in combination with our analysis of R expression indicates
an order of differentiation in which Gsc-ECD+ immature
cells give rise to Gsc+ECD+
R+ cells
that subsequently diverge to
Gsc+ECD+
R- and
Gsc+ECD-
R+ cells.
Nascent Gsc+ECD+ population contains mesendoderm with potential to give rise to endoderm and mesoderm
We next characterized in detail both ECD+ and ECD-
populations on day 5 by RT-PCR and immunohistochemical staining. RT-PCR
analysis showed that ECD+ population expressed claudin 6, which is
involved in tight junction of epithelium
(Sousa-Nunes et al., 2003),
and other endodermal markers such as Foxa2
(Ang et al., 1993
) and
Sox17 (Kanai-Azuma et al.,
2002
; Tam et al.,
2003
) (Fig. 3A). By
contrast, ECD- cells were expressing a set of mesodermal markers,
including ßR, Cad11 (Kimura
et al., 1995
) and Vegfr2
(Fig. 2C,
Fig. 3A). Two morphologically
different cell types were recognized in day 5 cultures: one composed of
epithelial sheets and the other containing dispersed cells
(Fig. 3B). Co-existence of
Gsc+ECD+Foxa2+ epithelial-like cells and
Gsc+ECD-Foxa2- non-epithelial-like cells in
the same culture was confirmed by fluorescent immunohistochemistry
(Fig. 3B).
The expression of Sox17 and the cytokeratins Krt2-8,
cytokeratin 18 and Krt119 (Owens
and Lane, 2003) was maintained during further culture of
Gsc+ECD+ population in SCM, whereas the expression of
Gsc and Mixl1, which is one of mesendoderm markers
(Hart et al., 2002
), was
successively downregulated and
-fetoprotein (Afp)
(Koike and Shiojiri, 1996
) was
newly expressed (Fig. 3C). To
further characterize Gsc+ECD+ cells in day 6 cultures,
we attempted to induce albumin-producing hepatocytes and pancreatic
ß-cells. The albumin-producing cells that were also expressing other
hepatic markers such as Hnf6
(Landry et al., 1997
;
Rausa et al., 1997
) could be
generated from Gsc+ECD+ cells
(Fig. 3D). These results
suggest that the Gsc+ECD+ population in our defined ES
cell culture contains precursors that can give rise to more mature endodermal
lineages. At present, however, we could not determine conditions for inducing
other endoderm-derived cells, such as pancreatic cells.
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To evaluate the differentiation potential of Gsc+ECD-
cells that diverge from nascent Gsc+ECD+ cells, day 6
Gsc+ECD- cells were sorted and cultured under selective
conditions for inducing mesodermal lineages such as osteoblasts and
endothelial cells. This ECD- population could give rise to
osteocytes and endothelial cells, which are typical progenies of mesoderm
(Fig. 4A). Though almost all of
Gsc+ECD- cells expressed Vegfr2, the efficiency
of endothelial differentiation from Gsc+ECD- population
was one-tenth that in Vegfr2+ lateral mesoderm cells [reported in
our previous study (Yamashita et al.,
2000) (data not shown)].
The above data suggest that the first Gsc+ECD+ population generated in ES cell differentiation culture represents presumptive mesendoderm with potential to give rise to both endoderm and mesoderm. This potential was vividly illustrated by immunofluorescent images of day 6.5 cultures in which Gsc+ECD+Foxa2+ cells were wedged between Gsc-ECD+Foxa2+ endoderm cells and Gsc+ECD-Foxa2- mesoderm cells (Fig. 4B).
Single cell assay for the bi-potent mesendodermal cells
Though the experiments described above suggest that the day 4
Gsc+ECD+ population contains mesendodermal cells that
can give rise to both endoderm and mesoderm, it is necessary to perform a
clonal analysis in order to prove the multipotency of an individual cell. To
obtain the efficient growth in single cell culture, we modified our culture
conditions into that including 10% FCS, LiCl, activin and basic Fgf, although
it is very difficult to judge whether these culture conditions are optimal for
this clonogenic assay.
Gsc+ECD+ cells from day 4 cultures were seeded by single cell deposition. Following a 3-day incubation, all wells were stained with anti-Foxa2 and a mixture of anti-Vegfr2 and anti-ßR mAbs to specify endoderm and mesoderm, respectively. The frequency of Gsc+ECD+ cells that undergo clonogenic growth was only about 0.5% under this condition (Table 1). From 64 wells in which we could observe more than three cells after staining, 45 contained only mesoderm cells and 19 contained both mesoderm and endoderm (Table 1; Fig. 4C). We could not detect any wells containing only endoderm cells. Co-existence of mesoderm cells may be required for the growth of endoderm. In conjunction with results of our mass culture of Gsc+ECD+ cells, this single cell assay demonstrates that bi-potent mesendodermal cells exist in our mouse ES cell culture.
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|
Purification of mesendoderm cells from genetically unmanipulated ES cell lines
As almost all of day 4 Gsc+ECD+ cells induced in our
defined culture condition co-express R,
ECD+
R+ cells in day 4 culture theoretically
correspond to Gsc+ECD+ population. In order to
investigate this possibility, we cultured unmodified EB5 ES cells under our
defined condition and ECD+
R+ cells were purified
from day 4 culture (Fig. 6A).
The sorted cells were further incubated for 2 days and ECD+
(population B in Fig. 6A) cells
were isolated for the analysis of endoderm marker expression and for the
establishment of endoderm cell lines.
A set of endodermal markers, including claudin 6, Foxa2 and
Sox17, was expressed in ECD+ population but not in
ECD- population, whereas mesoderm markers were expressed in
ECD- population (Fig.
6B). Moreover, we observed that Gsc was expressed in both
ECD+ and ECD- populations
(Fig. 6B). These results
indicate that ECD+ and ECD- population derived from day
4 ECD+R+ cells in genetically unmanipulated ES
cell cultures correspond to ECD+ and ECD- population in
day 6 Gsc+ population, respectively. Moreover, ECD+
endoderm cell lines have been established from unmanipulated ES cells by the
same protocol as that used for Gscgfp/+ ES cells
(Fig. 6C). These results
suggest that the method developed in this study is powerful for the
purification of mesendodermal cells in ES cell differentiation culture that
can give rise to both endodermal and mesodermal cells.
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Discussion |
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According to previous studies, Gsc is strongly expressed in EGO and MGO of
gastrula stage embryos (Belo et al.,
1998; Kinder et al.,
2001
). Gsc+ cells generated under our culture condition
express a set of organizer specific genes such as Foxa2, Lim1 and Chordin, as
well as T (a pan-mesoderm marker), and can give rise to both endodermal and
mesodermal cells. Although Gsc is also known to be expressed in some visceral
endoderm cells (Perea-Gomez et al.,
1999
), the expression of visceral markers such as Sox7
and Hnf4 are not induced in our defined culture condition. These
results suggest that ES cell-derived Gsc+ cells correspond to cells
in EGO and MGO that give rise to anterior endoderm, cranial mesenchyme and
somites (Kinder et al.,
2001
).
The defined condition together with surface markers exploited in this study
allowed us to delineate the differentiation course of definitive endoderm via
Gsc+ mesendoderm in vitro. Fig.
7 summarizes our understanding of the differentiation of
mesendoderm from ES cells, which is the first detailed model of the
differentiation course of mesendoderm. It is likely that mesendodermal cells
in EGO are ECD+ (Huber et al.,
1996), as it is a part of embryonic ectoderm. The expression of
ECD in epiblasts disappears along with their differentiation to mesoderm after
the exfoliation from the primitive streak. Thus, ECD is a useful marker for
distinguishing mesoderm from other germ layers. In our study, mesendoderm
cells diverge to Gsc+ECD+ endoderm and
Gsc+ECD- mesoderm precursors
(Fig. 7). The
Gsc+ECD+ endoderm precursors differentiate into
definitive endoderm, whereas Gsc+ECD- mesoderm
precursors can give rise to mesodermal lineages. Our previous observations
that both
R and Vegfr2 are expressed in anterior migrating mesoderm
cells agree with the surface phenotype of Gsc+ mesoderm cells in
culture (Kataoka et al.,
1997
). Thus, as is often the case with other cell processes such
as haematopoietic differentiation (Nakano
et al., 1996
), the process of mesendodermal differentiation, even
under such a selective culture condition, appears to correlate well with that
in the actual embryo.
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The fresh endoderm precursors defined by the expression of Gsc and ECD on
day 6 could give rise to HNF6+ albumin-producing cells. However, we
could not induce cells displaying pancreatic markers from
Gsc+ECD+ population, although we applied usual
pancreatic induction methods in which EB culture was used
(Hori et al., 2002;
Lumelsky et al., 2001
). We
speculate that unknown combination of growth factors or co-cultures with other
lineage cells are required for the differentiation from highly purified
Gsc+ECD+ cells to pancreatic cells. During long-term
propagation in vitro, the potential of differentiation into
albumin+ cells is lost and the culture is dominated by the cells
expressing molecules specific for immature endodermal lineage, such as Foxa2,
Sox17 and Gata4, but not markers for more mature stage, such as albumin and
Pdx1. Loss of differentiation potential into mature endoderm in this cell line
may result from other processes such as overdomination of a particular cell
type. Our result shows for the first time that some of ES cell-derived
endoderm cells bear an ability to undergo sustained proliferation in vitro.
This ability is not due to the malignant transformation, because the tumour
formations were not observed by transplantation into immunodeficient mice. To
our disappointment, however, we have no evidence that these cell lines can
undergo further differentiation into mature endodermal cells such as
pancreatic ß-cells. Further studies are necessary to determine whether
endoderm cells from the actual embryo has a similar ability to undergo
sustained proliferation or whether such a high proliferative activity is
specific to ES cell-derived endoderm cells.
Guided differentiation of ES cells by defined culture condition
One of ultimate goals for in vitro ES cell differentiation is to prepare a
sufficient number of pure cells by controlling ES cell differentiation. There
are thought to be two ways to achieve this. One is to consider that ES cell
differentiation requires the environments which are similar to those present
in the actual embryo. The most typical example of such a method is EB culture
system for ES cell differentiation (Burkert
et al., 1991). The other is based on the idea that steering ES
cell differentiation is attained only by the highly selective culture
condition that excludes differentiation into unnecessary lineages. In this
study, we sought tested the latter possibility, and have succeeded in
determining a defined condition that can generate almost pure Gsc+
population without using EB formation method. We have demonstrated that EB
culture, even using the same defined culture condition, is less efficient in
inducing Gsc+ cells than the two-dimensional (2D) culture on
collagen IV-coated dishes. This result indicates an inherent limitation of EB
system in guiding ES cell differentiation, as uncontrollable complexity is
inevitably associated with three-dimensional architecture in EB.
We realize that what is taking place in ES cell differentiation culture
under a defined condition does not necessarily recapitulate the
differentiation process of the actual embryo. However, it is also clear that
in vitro differentiation, even though induced under a highly artificial
condition, proceeds under similar constraints to that in the differentiation
process of the actual embryo. In this study, we have shown that nodal and
activin can selectively induce ES cell-derived Gsc+ mesendoderm.
This result is consistent with previous reports showing that nodal is an
indispensable molecule for inducing the organizer in both Xenopus and
mouse (Varlet et al., 1997;
Zhou et al., 1993
). Mutant
analyses of nodal-related genes in zebrafish and Xenopus have
indicated that they are required for the generation of both endoderm and
mesoderm (Osada and Wright,
1999
; Rodaway et al.,
1999
). Moreover, it has been shown that Nodal signalling has an
essential role in specification of anterior definitive endoderm and a part of
mesoderm in mouse (Lowe et al.,
2001
; Vincent et al.,
2003
). Our observation that Activin/nodal signal can induce
endoderm and mesoderm differentiation in ES cell culture is consistent with
these in vivo studies. In the actual embryo, however, it has been difficult to
specify at cellular level which differentiation process is regulated by nodal
or nodal-related molecules. In this sense, our method of inducing pure
Gsc+ population in ES cell cultures should complement previous in
vivo studies and provide a clearer view on the process of mesendoderm
differentiation.
In this study, we also aimed at developing a method to define mesendoderm
without using genetically manipulated ES cells. We have shown here that
mesendoderm is defined as ECD+R+ when these ES
cells are cultured under the selective culture condition containing activin.
As mesendoderm is one of the precursors of definitive endoderm
(Lu et al., 2001
;
Martinez Barbera et al.,
2000
), isolation and characterization of mesendodermal cells are
essential steps for generating more mature cells of definitive endodermal cell
lineage. We validated this method by establishing endoderm cell lines from
genetically unmanipulated ES cells. Indeed, this experimental system allowed
us to establish endoderm cell lines that are similar to those established from
Gscgfp/+ ES cell lines. Moreover, pure mesendoderm
population is also useful for screening defined culture conditions required
for subsequent differentiation steps towards mature endoderm cells. The trials
on this line are currently in progress.
The demonstration that activin can induce Gsc and R in ES cell
culture is consistent with previous studies that activin induced the
expression of both Gsc and
R in Xenopus animal cap cells
(Gurdon et al., 1995
;
Jones et al., 1993
;
Symes et al., 1994
). This
similarity between evolutionally distant species suggests that our defined
culture system, which can induce pure Gsc+ mesendoderm from mouse
ES cells, is also likely to be applicable to differentiation of human ES
cells.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/132/19/4363/DC1
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ACKNOWLEDGMENTS |
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