1 The Carl C. Icahn Center for Gene Therapy and Molecular Medicine, Mount Sinai
School of Medicine, New York, NY 10029, USA
2 Division of Pulmonary Biology, Children's Hospital Medical Center, Cincinnati,
OH 45229, USA
3 Department of Immunology, Medical Faculty/University Clinics, Ulm,
Germany
* Present address: Department of Public Health, Nara Medical University, Nara
634-8521, Japan
Present address: Paterson Institute for Cancer Research, Christie Hospital NHS
Trust, Wilmslow Road, Manchester M20 4BX, UK
Author for correspondence (e-mail:
gordon.keller{at}mssm.edu)
Accepted 3 December 2003
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SUMMARY |
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Key words: Stem cells, Endoderm
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Introduction |
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The close developmental association between mesoderm and endoderm, possibly
through a common progenitor, suggests that the same mechanisms regulate the
early induction events leading to the establishment of these lineages.
Although many factors and cellular interactions have been implicated in the
regulation of different stages of endoderm and mesoderm formation, a consensus
of findings from different studies point to an essential role for members of
the TGFß family of molecules at the earliest stages in this process
(Smith, 1993;
Hogan, 1996
;
Schier and Shen, 2000
;
Stainier, 2002
). Two of the
most notable factors in this regard are activin and nodal. Activin was
identified as a potential regulator of these early developmental decisions
based on its capacity to induce mesoderm and endoderm in Xenopus
animal caps in vitro (Smith et al.,
1990
; Jones et al.,
1993
; Gamer and Wright,
1995
; Ninomiya et al.,
1999
) and from the findings that interference with its receptor
function inhibited the development of these germ cell layers in the embryo
(Hemmati-Brivanlou and Melton,
1992
; Hemmati-Brivanlou and
Melton, 1994
). Nodal and related factors are expressed prior to
and during the onset of gastrulation and have been shown to play pivotal roles
at the earliest stages of mesoderm and endoderm development in the mouse,
Xenopus and zebrafish embryo
(Zhou et al., 1993
;
Conlon et al., 1994
;
Jones et al., 1995
;
Feldman et al., 1998
;
Osada and Wright, 1999
;
Gritsman et al., 2000
;
Lowe et al., 2001
).
Signaling events in the early embryo initiate the activation of a cascade
of transcription factors that function at different stages in the induction
and specification of definitive endoderm. With respect to the earliest
induction steps, studies in Xenopus and zebrafish have clearly
demonstrated a role for factors such as the homeodomain protein Mixer/Mix.3,
(Henry and Melton, 1998) the
Sry-related HMG-box transcription factor Sox 17
(Hudson et al., 1997
) and the
zinc-finger transcription factor Gata5
(Reiter et al., 1999
;
Reiter et al., 2001
;
Weber et al., 2000
). Targeting
experiments in the mouse have similarly shown that Sox17
(Kanai-Azuma et al., 2002
) and
the mix-like gene, Mixl-1 (Hart et al.,
2002
) are essential for endoderm development, indicating that the
mechanisms regulating early induction events are evolutionarily conserved.
Hepatocyte nuclear factors (HNFs), a group of proteins originally identified
as regulators of liver gene expression, also play important roles in endoderm
development (Darlington, 1999
).
In the mouse, Foxa2 (previously known as HNF3ß), a factor expressed in
the anterior region of the primitive streak, in endoderm and the early liver
(Monaghan et al., 1993
;
Sasaki and Hogan, 1993
), is
essential for the development of prospective foregut and midgut endoderm
(Ang et al., 1993
;
Weinstein et al., 1994
).
Beyond the induction stage, numerous other transcription factors are required
for endoderm patterning and organ development. Nkx2.1 is required for thyroid
development and lung morphogenesis
(Lazzaro et al., 1991
), Hhex
for liver and thyroid development
(Martinez Barbera et al.,
2000
), and Ipf1 (Pdx1) for the formation of the ventral and dorsal
pancreas (Jonsson et al.,
1994
).
Much of our current knowledge of endoderm induction is based on findings
from studies using model systems such as Xenopus and zebrafish that
provide easy access to early embryonic stages of development at a time when
lineage commitment decisions are taking place. By contrast, the mouse embryo
is much less amenable to such experimental approaches because of its difficult
accessibility and limiting amounts of tissue. The in vitro differentiation of
embryonic stem (ES) cells provides an attractive alternative model system to
the mouse embryo for addressing questions relating to early lineage commitment
(Keller, 1995;
Smith, 2001
). Under
appropriate culture conditions, ES cells will differentiate into embryoid
bodies (EBs) that can contain derivatives of all three germ cell layers. To
date, the majority of such studies have focused on the development of mesoderm
and ectoderm derivatives and as a consequence, conditions have been
established for the efficient and reproducible differentiation of the
hematopoietic, vascular, muscle and neural lineages
(Keller et al., 1993
;
Nakano et al., 1994
;
Rohwedel et al., 1994
;
Bain et al., 1995
;
Okabe et al., 1996
;
Vittet et al., 1996
;
Nishikawa et al., 1998
;
Czyz and Wobus, 2001
). More
recently, several studies have provided evidence for endoderm development in
ES differentiation cultures and demonstrated the generation of
insulin-expressing cells and cells with hepatocyte characteristics
(Abe et al., 1996
;
Hamazaki et al., 2001
;
Lumelsky et al., 2001
;
Hori et al., 2002
;
Jones et al., 2002
;
Yamada et al., 2002a
;
Yamada et al., 2002b
;
Blyszczuk et al., 2003
). None,
however, has established conditions for the efficient induction of endoderm,
nor has any defined the origin of the specific cell populations in the
study.
In this report, we have investigated the events that regulate endoderm
development in ES differentiation cultures and demonstrate that exposure of
EBs to factor(s) present in serum for restricted periods of time is essential
for the establishment of this lineage. In addition to serum factors, we show
that activin A can induce mesoderm and endoderm formation in EBs in serum-free
cultures and that lineage development is dependent on the concentration of
factor used. Using an ES cell line with the GFP cDNA targeted to the brachyury
locus (Fehling et al., 2003),
we provide evidence that the endoderm lineage develops from a
brachyury+ population with mesoderm potential.
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Materials and methods |
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For endoderm differentiation, serum-stimulated EBs were harvested at day 2.5 of differentiation, allowed to settle by gravity and then replated in 60 mm dishes in IMDM supplemented with 15% serum replacement (SR; Gibco/BRL), penicillin/streptomycin, 2 mM glutamine, 0.5 mM ascorbic acid and 4.5x104 M MTG (hereafter referred to as serum replacement medium). Activin induction was carried out using a two-step culture protocol. In the first step, EB differentiation was initiated in Stem Pro 34 medium (Gibco) supplemented with 2 mM glutamine, 0.5 mM ascorbic acid, 4.5x104 M MTG and Kit ligand (1% conditioned medium) at a concentration of 2x103 ES cells/ml. For the second step, EBs were harvested at 48 hours of differentiation, allowed to settle by gravity in a 50 ml tube and transferred to new dishes and cultured in IMDM supplemented with 15% SR, 2 mM glutamine, 0.5 mM ascorbic acid, 4.5x104 M MTG and different concentrations of human activin A (R&D Systems).
Hepatocyte differentiation
For the hepatocyte differentiation, day 2.5 serum-induced EBs were
transferred to serum replacement media for an additional 3.5 to 7.5 days. At
days 6 or 10 of differentiation, EBs were harvested and replated intact on
six-well tissue culture dishes coated with matrigel (Becton Dickenson, San
Jose, CA) (20 EBs per well) in IMDM with 15% FCS and
107 M dexamethasone (Dex) (Sigma) (referred to as serum
hepatocyte cultures). Cells were harvested for expression analysis at day 14
(total time) of culture. For the analysis of brachyury subpopulations,
GFP-Bry+ and GFP-Bry cells were sorted at day
2.5, and reaggregated (3x105 cells/ml) for 1 day in ultra low
attachment 24-well plates (Costar) in serum replacement media. The
reaggregated EBs were transferred to 60 mm petri-grade dishes with serum
replacement media. At a total of 6 or 10 days of differentiation, EBs were
replated on matrigel coated 6 well dishes in serum replacement media
supplemented with 5 ng/ml bFGF (R&D Systems, Minneapolis, MN) (serum-free
hepatocyte conditions). Cells from the replated cultures were harvested at day
14 (total time for differentiation) for RNA isolation and immunostaining. For
activin-induced cultures, cells from the pre-sorted, the GFP-Bry+
and GFP-Bry populations from day five EBs were reaggregated
in serum replacement media and then cultured in the same media in the absence
of activin for 8 days. At day 13, the reaggregated EBs were replated in serum
hepatocyte conditions for four days and then harvested for RT-PCR
analysis.
Hematopoietic progenitor assays
For hematopoietic colony assays, EBs were trypsinized to single cell
suspensions and plated (5x1041x105
cells/ml) in 1% methylcellulose containing 10% plasma derived serum (Antech,
Tyler, TX), 5% PFHM-II and the following cytokines; Kit ligand (KL, 1%
conditioned medium), interleukin 3 (IL3, 1% conditioned medium),
thrombopoietin (Tpo, 5 ng/ml), erythropoietin (Epo, 2 U/ml), interleukin-11
(IL-11, 5 ng/ml), granulocyte-macrophage colony stimulating factor (GM-CSF, 3
ng/ml) and macrophage colony-stimulating factor (M-CSF, 5 ng/ml). Primitive
erythroid colonies were counted at day 4, whereas macrophage and multilineage
colonies were counted at day 7 of culture. Kit ligand was derived from media
conditioned by CHO cells transfected with a KL expression vector (kindly
provided by Genetics Institute). IL3 was obtained from medium conditioned by
X63 AG8-653 myeloma cells transfected with a vector expressing IL3
(Karasuyama and Melchers,
1988); GM-CSF, M-CSF and TPO were purchased from R&D
Systems.
Gene expression analysis
Total RNA was extracted using a RNeasy mini-kit and treated with RNase free
DNase (Qiagen, Valencia, CA). Total RNA (2 µg) was reverse-transcribed into
cDNA with random hexamers using Omniscript RT kit (Qiagen). PCR was performed
with Taq polymerase (Promega, Madison, WI) or platinum Taq (Invitrogen,
Carlsbad, CA) in PCR buffer, 2.5 mM, 0.2 µM dNTPs. Cycling conditions were
as follows; 94°C for 5 minutes followed by 25-40 cycles of amplification
(94°C denaturation for 1 minute, annealing for 30 seconds, 72°C
elongation for 1 minute), with a final incubation at 72°C for 7 minutes.
Details of primer sequences, annealing temperature and cycle numbers for each
PCR reaction are shown in Table
1.
|
Immunostaining
Foxa2 staining of brachyury+ cells was carried out in microtiter
wells. Cells (1x105 ml) were centrifuged in 96-well plates,
the supernatant removed and the cells then fixed for 30 minutes in 100 µl
of paraformaldehyde solution (4%). After fixation, the cells were washed twice
with PBS, permeabilized in PBS containing 0.2% triton for 5 minutes at room
temperature, washed with 150 µl of PBS with 0.1% tween 20 and 0.02% azide
and then blocked with PBS containing 10% horse serum for 10 minutes at room
temperature. Following the blocking step, the cells were washed again with PBS
with 0.1% tween (wash media) and then incubated with an anti-Foxa2 antibody
(goat polyclonal P-19, Santa Cruz) in PBS containing 10% FCS and 0.1% tween
(staining media) for 30 minutes at room temperature. The stained cells were
washed and then incubated with a Cy3-labeled antigoat IgG (Jackson
Immunoresearch, West Grove, PA) in staining solution for 30 minutes at room
temperature. After the second staining step, the cells were washed and then
covered with 10 µl of vectastain mounting media containing DAPI. The cells
in the mounting media were transferred to a slide and covered with a
coverslip. For the albumin staining, day 10 EBs were partially dissociated to
small aggregates by a 30-second trypsin treatment and cultured on HCl-treated,
gelatin-coated glass cover slips for 4 days. For skeletal muscle staining,
intact 10-day-old activin stimulated EBs were cultured on coverslips for 4
days. After culture, the cells were processes as described above for the Foxa2
staining. Fixed and blocked cells were incubated for 1 hour with either
anti-albumin (Biogenesis, Kingston, NH), anti-skeletal myosin or
-actinin (Sigma) primary antibodies. Expression of these proteins was
visualized using a Cy3-conjugated anti-mouse or rabbit IgG secondary antibody
(Jackson Immunoresearch, West Grove, PA). Tissue sections were stained with
either an anti-Foxa2 (Lake Placid, New York) or anti-Ifabp
(Green et al., 1992b
) primary
antibody. Secondary antibody treatment and color development were carried out
using Vectastain ABC kits (Vector Laboratory Burlingame California).
In situ hybridization
The murine cDNA for Sftpc (758 bp) was used as a template for generating
riboprobes. In situ hybridization was performed on paraformaldehyde fixed,
paraffin wax-embedded sections as previously described
(Deterding and Shannon, 1995)
with the exception that 33P-UTP was used for labeling the
probe.
Transplantation of EBs under kidney capsule
Cell aggregates were harvested from the matrigel cultures by cell scraping
and then transplanted under the kidney capsule of 5-weekold female SCID-beige
mice. Three to four weeks following transplantation, the recipients were
sacrificed, the kidneys removed and fixed in 4% paraformaldehyde. After
fixation, kidneys were embedded in paraffin wax and sectioned for H/E and
D-PAS staining, for immunostaining and for in situ hybridization.
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Results |
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Development of the hepatocyte lineage from EBs expressing endoderm genes
Previous studies have demonstrated that ES cells can generate cells with
hepatocyte characteristics indicating that derivatives of definitive endoderm
can develop in this model system (Hamazaki
et al., 2001; Jones et al.,
2002
; Yamada et al.,
2002a
). To determine if the EBs generated using the
serum/serum-free protocol could differentiate to hepatocyte-like cells, they
were subsequently cultured under conditions known to support development of
this lineage (Hamazaki et al.,
2001
; Kamiya et al.,
2001
; Kamiya et al.,
2002
). Early hepatocyte development was marked by the presence of
-fetoprotien (AFP), albumin (ALB1), transthyretin (TTR) and
alpha1-antitrypsin (AAT; SERPINA1Mouse Genome Informatics) expression,
while further maturation of the lineage was defined by expression of tyrosine
aminotransferase (TAT) and carbamoyl phosphate synthetase I (CPS1), genes that
encode enzymes found in mature hepatocytes
(Hamazaki et al., 2001
;
Yamada et al., 2002a
). When
10-day-old EBs (2.5 days serum-containing medium, 7.5 days SF) were replated
on matrigel-coated plates in cultures containing serum and dexamethasone for 4
days (serum hepatocyte conditions), expression of all genes could be detected,
indicating differentiation to the hepatocyte lineage
(Fig. 1D, S/SF). Additional
manipulations of the culture conditions revealed that EBs could be replated on
matrigel at 6 rather than 10 days of differentiation and that the replated
cultures could also be maintained in serum free conditions with bFGF
(serum-free hepatocyte conditions) (not shown). The expression of genes
associated with hepatocyte development and maturation in cells from these
replated cultures is a strong indication that definitive endoderm is induced
in EBs generated by a 2.5-day exposure to serum. Control cultures of EBs
maintained in serum for the entire 10-day period prior to replating did not
give rise to cell populations that expressed any of these hepatocyte genes,
suggesting that endoderm potential is not maintained under a continuous serum
stimulus (Fig. 1D,S).
Endoderm develops from brachyury+ cells
To define the relationship between cells with endodermal characteristics
and mesoderm in the ES/EB system, brachyury+ (GFP-Bry+)
and brachyury (GFP-Bry) cells were
isolated from day 2.5 serum-stimulated EBs on the basis of GFP expression and
assayed for endoderm potential (Fig.
2A). Cells from each fraction were allowed to reaggregate for 24
hours in serum-free media, as we have recently demonstrated that such
aggregates will continue to differentiate with further culturing
(Fehling et al., 2003). The
aggregates from each fraction were cultured for an additional 2.5 days in
serum-free conditions (total of 6 days of differentiation) and then replated
for 4 days in serum-free hepatocyte conditions
(Fig. 2A). Expression analysis
of various endoderm genes was performed on the initial GFP-Bry sorted
populations, the day 6 EBs and the replated cultures. Foxa2 and
Mixl1 were expressed in the day 2.5 EBs and this expression
segregated to the brachyury+ fraction
(Fig. 2B; d2.5). Sox17
and Hhex expression in the day 6 EBs
(Fig. 2B; d6) and in the day 10
replated cultures (Fig. 2B; d10) as well as Afp and Alb1 expression in the replated
cultures was also restricted to cell populations derived from the
brachyury+ cells. By contrast, Pax6 and Neurod1
(Lee et al., 1995
), genes
associated with neuroectoderm development were expressed in the
brachyury-derived cell populations. These findings indicate
that endoderm develops from a brachyury+ population, whereas
neuroectoderm derives from brachyury cells.
|
Activin A induces mesoderm and endoderm in EBs
To define the regulation of endoderm induction in the EB model in more
detail, we next attempted to replace the serum-stimulus with specific factors.
As activin has been shown to have both mesoderm and endoderm-inducing
potential in the Xenopus model
(Smith et al., 1990;
Jones et al., 1993
;
Gamer and Wright, 1995
;
Ninomiya et al., 1999
), we
tested it for its ability to exert these effects in the EB system in the
absence of serum. Activin induced brachyury expression to almost the same
extent observed with serum induction (Fig.
3A), although the kinetics of this expression pattern was altered,
being delayed by
48 hours (compare
Fig. 3A with
Fig. 1B). Molecular analysis of
the activin-induced EBs demonstrated the expression of Foxa2 and
Mixl1 by day 5 of differentiation, Sox17, Hhex and
Hnf4 (Duncan et al.,
1994
) by day 6 and Ipf1 by day 7. Gata1 was not
expressed at any of the time points in the activin stimulated EBs, although
low levels of Kdr (Flk1) were found at days 6 and 7 of
differentiation. None of these genes was expressed in EBs differentiated in
the absence of activin (activin). Pax6 displayed an inverse
pattern of expression and was present in the EBs generated in the absence of
activin, but not in those differentiated in its presence. These findings
indicate that, at the dose used, activin can stimulate brachyury expression
and endoderm differentiation in EBs in the absence of serum.
|
Mesoderm and endoderm potential of activin-induced cell populations
As expected from the lack of Gata1 expression, no hematopoietic
progenitors were detected in day 5 activin-treated serum-free EBs
(Fig. 4A, transfer). To
determine if the activin-induced brachyury+ population contained
mesoderm with hematopoietic potential, day 5 or 6 EBs stimulated with
different concentrations of activin were transferred to serum containing
medium for 3 days and then assayed for hematopoietic progenitors. Day 5 EBs
differentiated in the presence of 3 or 100 ng of activin generated primitive
and definitive hematopoietic progenitors following the 3-day exposure to serum
(Fig. 4A, +transfer).
Significantly lower numbers of progenitors were present in activin-stimulated
day 6 EBs and none was detected in day 5 EBs initiated in the absence of
activin. The development of hematopoietic progenitors in EBs stimulated with 3
and 100 ng/ml of activin demonstrates that both low and high concentrations of
this factor can induce mesoderm with hematopoietic potential. The reduced
hematopoietic activity in day 6 EBs suggests that this potential is
transient.
|
As a final characterization of the activin-stimulated cells, brachyury+ and brachyury populations isolated from EBs stimulated with low and high concentrations of factor were reaggregated and cultured as described and then analyzed for expression of the skeletal muscle and endoderm genes. As shown in Fig. 4D, both Myf5 and skeletal actin expression was restricted to the population generated from the brachyury+ population isolated from EBs stimulated with 3 ng/ml of activin. Similarly, the endoderm genes were expressed in the brachyury+-derived cells isolated from EBs generated in the presence of 100 ng/ml of factor. These findings further support the concept that both mesoderm and endoderm develop from a brachyury+ population.
In vivo potential of activin-induced GFP-Bry+ populations
To evaluate the developmental potential of the activin-induced cells in
vivo, cells from the GFP-Bry+ and GFP-Bry
fractions isolated from day 5 EBs induced with 100 ng/ml of factor were
reaggregated and cultured as EBs for 8 days (serum free), replated in serum
hepatocyte conditions for 4 days and then transplanted under the kidney
capsules of SCID-beige mice. The extended culture time as EBs was included to
promote maturation of the endoderm populations. Three weeks after
transplantation, the mice were sacrificed and the kidneys analyzed. Grafts
from the GFP-Bry+ population were relatively small and homogenous
in sized and their growth was restricted to only part of the kidney
(Fig. 5A). By contrast, grafts
from the GFP-Bry fraction were very large, 100 times
the size of those from the GFP-Bry+ cells, heterogeneous in
appearance and often engulfed the entire kidney
(Fig. 5A). Histological
analysis indicated that the GFP-Bry+ grafts contained both endoderm
and mesoderm derivatives. Endoderm was represented predominantly by the
presence of ductal structures that consisted of cells with the morphology of
gut epithelial cells (Gu) and tall columnar cells (C), possibly representing
bronchial epithelium (Fig. 5B). Cells with the morphology of hepatocytes were not detected in these grafts.
With respect to mesoderm derivatives, skeletal muscle (SM) and adipocyte
tissue were detected and in some instances bone (B) was also present. The
cells within these structures appeared to be well differentiated, indicating
the presence of mature populations. By contrast, grafts from the
GFP-Bry cells displayed morphological characteristics of ES
cell-derived teratomas and consisted of derivatives of all three germ layers.
The GFP-Bry derived grafts contained substantial populations
of neural tissue (brain; Br), skeletal muscle, gut-like epithelial cells as
well as many regions of immature cells, including neural tube-like structures
(NT) that were not found in the grafts from the GFP-Bry+ cells.
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Discussion |
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A comparison of endoderm development in serum-containing and serum-free
cultures highlights the importance of providing appropriate inducing factors
for the generation of differentiated progeny from ES cells. Although both
protocols result in the generation of endoderm, the extent of endoderm
induction within the EBs, based on Foxa2 protein analysis, appears to be
significantly higher in the serum-free/activin cultures. In addition, activin
consistently induced the expression of Sftpc and Ipf1, genes
indicative of lung and pancreas specification that were seldom detected using
our serum-induction protocol. The upregulation of Ipf1 following
activin treatment suggests that this protocol may provide a novel approach for
the generation of pancreatic ß cells from differentiating ES cells. A
number of recent studies have described the development of insulin-expressing
cells in ES differentiation cultures
(Lumelsky et al., 2001;
Hori et al., 2002
;
Blyszczuk et al., 2003
).
However, the interpretation of these studies is complicated by the fact that
the analysis was carried out in mixed lineage cultures. The ability to
separate neuroectoderm from mesoderm and endoderm by brachyury expression will
enable us to define the origin of the Ipf1-expressing cells in the
developing EBs, and ultimately follow their differentiation to a ß cell
fate.
The mesoderm potential induced by activin and serum also differed. A 6-day
exposure to serum induced hematopoietic progenitor development
(Fig. 1C), but little if any
skeletal muscle potential (A.K. and G.K., unpublished). By contrast, activin
induced both skeletal muscle and hematopoietic mesoderm development. However,
unlike the serum-induced hematopoietic program that resulted in the generation
of progenitors, the program induced by activin appears to consist of
hematopoietic mesoderm that has not yet advanced to the progenitor stage.
Progression to the progenitor stage of development was observed following a
3-day culture period in serum. Previous studies have demonstrated the
generation of skeletal muscle from ES cells differentiated in serum-containing
cultures (Rohwedel et al.,
1994). Our activin induction protocol represents a significant
advancement over these early studies in that it is faster and more efficient.
The combination of activin induction with the isolation of
brachyury+ populations provides a novel approach for the generation
of large numbers of skeletal myocytes for cell replacement studies.
The demonstration that activin can induce mesoderm and endoderm
differentiation in the ES cultures in the absence of serum is consistent with
numerous studies that have defined a requirement for factors of the TGFß
family in the early induction of these lineages
(Smith, 1993;
Gurdon et al., 1994
;
Schier and Shen, 2000
).
Although it is well established that activin can induce mesoderm and endoderm
in different model systems, targeting studies in mice would suggest that it is
not the endogenous factor that regulates these developmental decisions in the
early embryo (Vassalli et al.,
1994
; Matzuk et al.,
1995
). Rather, most evidence suggests that nodal and nodal-like
factors function in the capacity to regulate early mesoderm and endoderm
development in vivo (Schier and Shen,
2000
; Whitman,
2001
). In a recent study, we have shown that nodal is expressed in
GFP-Bry+ populations isolated from early EBs, suggesting that it
may also be regulating lineage fates in this model
(Fehling et al., 2003
). The
overlapping activities of activin and nodal early in development may reflect
the fact that they can bind the same receptors and thus initiate the same
signaling events (Schier and Shen,
2000
).
The response to varying concentrations of activin in the ES system is also
consistent with studies in Xenopus that have demonstrated that
different concentrations of activin will induce different fates in animal cap
cells in culture (Green and Smith,
1990; Green et al.,
1992a
; Jones et al.,
1993
; Gamer and Wright,
1995
; McDowell et al.,
1997
; Ninomiya et al.,
1999
). In the Xenopus model, high concentrations of this
factor were shown to induce dorsal mesoderm and endoderm, whereas low
concentrations induce ventral mesoderm. In the ES/EB model, endoderm was
induced most efficiently at high concentrations, whereas low and intermediate
levels of activin induced mesoderm. The differential response to activin in
the ES/EB model is consistent with its functioning as a morphogen in which
different concentrations can induce different fates in a given cell.
Alternatively, different concentrations of activin could be stimulating
distinct subpopulations of progenitors, those with endoderm potential
requiring a higher activin concentration than those with mesoderm potential.
Distinguishing between these possibilities will require the establishment of
conditions that will enable the analysis of the fate of single cells following
induction.
The observation that endoderm derives from GFP-Bry+ cells
suggests that this germ layer develops from a bi-potential mesendoderm
population that co-expressed brachyury and Foxa2. This ES-derived GFP-Bry
population could be similar to cells in the anterior region of the primitive
streak of the mouse embryo that express these genes
(Wilkinson et al., 1990;
Monaghan et al., 1993
;
Sasaki and Hogan, 1993
) and
give rise to the first endodermal cells
(Wells and Melton, 1999
).
Studies in other species have identified populations with mesendoderm
potential and have provided evidence that although most endoderm is derived
from this population, only a subset of mesoderm, including that fated to blood
and cardiac muscle arises from these precursors
(Rodaway and Patient, 2001
).
In this study, we demonstrated that the skeletal muscle lineage develops from
an activin-induced brachyury+ population and in a recent report we
showed that serum-induced brachyury+ cells isolated at the same
stage of development display hematopoietic and endothelial potential
(Fehling et al., 2003
). In
addition to these lineages, beating cardiac myocytes were often found in the
cultures of brachyury+ cells maintained in hepatocyte
differentiation conditions (A.K. and G.K., unpublished). Which, if any, of
these mesoderm lineages shares a common precursor with endoderm will require
clonal analysis. Access to the brachyury+ cells from the EBs will
enable us to not only address this question, but also define and characterize
the regulatory mechanisms involved in the induction, patterning and
tissue-specific differentiation of endoderm and mesoderm.
The findings from the kidney capsule transplantation experiment further
support our in vitro studies and demonstrate that the activin-induced EB cells
displayed endoderm and mesoderm potential in vivo. The identification of
Sftpc- and Ifabp-expressing cells in the grafts clearly demonstrates the
potential of these cells to generated endoderm-derived tissues. Although we
could reproducibly generate hepatocyte-like cells in culture, this lineage did
not persist or expand in the grafts under the kidney capsule. The lack of
hepatocyte differentiation may indicate that the environment of the kidney
capsule is not optimal for the differentiation and growth of this lineage.
Ultimately, functional analysis of the ES cell-derived hepatocyte-like cells
will require transplantation into animal models of liver failure, such as the
fumarylacetoacetate hydrolase (FAH)-deficient mouse
(Grompe et al., 1993). The
development of teratomas from the brachyury fraction is
consistent with our previous findings demonstrating that undifferentiated ES
cells segregate to the negative population
(Fehling et al., 2003
). The
fact that teratomas developed from the GFP-Bry cells even
after extensive time in culture, highlights the importance of establishing
methods for removing residual ES cells from the cultured population prior to
engraftment. Isolation of brachyury+ cells prior to culture
significantly reduced the development of multilineage teratomas.
In summary, the findings reported here demonstrate that ES cells can generate definitive endoderm-derived cell populations under defined conditions in culture. Access to activin-induced brachyury+ cells provides a unique population of enriched endoderm progenitors for future studies aimed at their tissue specific differentiation, as well as the identification and characterization of genes involved in these processes. Finally, defining the events involved in the development of endoderm from mouse ES cells in this study represents an important first step in generating similar populations from human ES cells.
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ACKNOWLEDGMENTS |
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