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Efficient differentiation of human embryonic stem cells
to definitive endoderm
Kevin A D’Amour, Alan D Agulnick, Susan Eliazer, Olivia G Kelly, Evert Kroon & Emmanuel E Baetge
The potential of human embryonic stem (hES) cells to differentiate into cell types of a variety of organs has generated much
excitement over the possible use of hES cells in therapeutic applications. Of great interest are organs derived from definitive
endoderm, such as the pancreas. We have focused on directing hES cells to the definitive endoderm lineage as this step is a
prerequisite for efficient differentiation to mature endoderm derivatives. Differentiation of hES cells in the presence of activin
A and low serum produced cultures consisting of up to 80% definitive endoderm cells. This population was further enriched to
near homogeneity using the cell-surface receptor CXCR4. The process of definitive endoderm formation in differentiating hES cell
cultures includes an apparent epithelial-to-mesenchymal transition and a dynamic gene expression profile that are reminiscent
of vertebrate gastrulation. These findings may facilitate the use of hES cells for therapeutic purposes and as in vitro models of
development.
The definitive endoderm gives rise to the epithelial lining of the respiratory and digestive tracts and to the thyroid, thymus, lungs, liver and pancreas. It is formed during gastrulation, in which pluripotent epiblast cells
are allocated to the three principal germ layers—ectoderm, mesoderm
and definitive endoderm1,2. The initiation of this process is evidenced
by the appearance of the primitive streak in the posterior epiblast. As
epiblast cells ingress through the primitive streak they undergo an epithelial-to-mesenchymal transition (EMT) and become either mesoderm
or definitive endoderm3. Although it has been suggested that these two
cell types arise from a common precursor in the primitive streak, called
the mesendoderm, the existence of a single embryonic cell with bipotent
properties has not been proven in mammals.
Genetic analyses in mice have shown that disruption of either the
WNT or TGFβ signaling pathways prevents formation of the primitive streak, the mesoderm and the definitive endoderm4–8. In addition,
Nodal, a member of the TGFβ superfamily, is essential for specification
of endoderm during gastrulation in mice with high levels of Nodal
signaling specifying definitive endoderm and lower levels of Nodal signaling specifying mesoderm9,10. Although Nodal is an attractive candidate for inducing definitive endoderm differentiation of hES cells, a
source of highly active protein is not readily available. However, another
TGFβ family member, activin, binds the same receptors as Nodal (with
the exception of the coreceptor cripto), triggering similar intracellular
signaling events11, and therefore can be used to mimic Nodal activity
in vitro.
Many genes expressed in definitive endoderm are also expressed
in other embryonic tissues, including mesoderm and neurectoderm,
and in extra-embryonic tissues such as the primitive endoderm and its
derivatives, the visceral endoderm and parietal endoderm. Definitive
endoderm arising within a complex culture can therefore be identified
1CyThera
only by measuring the gene expression of multiple markers of target
and nontarget cell types. Furthermore, studies of gene expression at the
population level must be validated at the single-cell level to determine
the efficiency of definitive endoderm production. A list of genes useful
for determining lineage allocation in the gastrulation-stage embryo is
provided in Supplementary Table 1 online.
In a recent study, mouse ES cells were differentiated to mesendoderm
and subsequently to mesoderm and definitive endoderm by treatment
with activin12. However, the efficiency of definitive endoderm production was unclear because the principal marker used at the single-cell level
was Foxa2, which is also expressed in axial mesoderm13–15.
Here we describe the production of enriched cultures of hES cell–
derived definitive endoderm in the presence of activin A and low serum.
By virtue of multiple markers analyzed, we show that we have made
definitive endoderm and not primitive endoderm. Our data also indicate that the temporal sequence of gene expression characteristic of
hES cell differentiation to definitive endoderm is similar to the transitions that occur in the course of definitive endoderm differentiation
during vertebrate gastrulation. Transplantation of these cells under the
kidney capsule resulted in their differentiation into more mature cells
of endodermal organs. Finally, hES cell–derived definitive endoderm
cells were purified to near homogeneity using the cell-surface receptor
CXCR4 (ref. 16).
RESULTS
Serum concentration affects definitive endoderm production
Because Nodal/activin induces endoderm in vertebrates17, we tested the
effects of activin A on human ES cells. Lowering the serum concentration in the presence of 100 ng/ml activin A for 5 d resulted in maximally
elevated expression of genes expressed in vertebrate definitive endoderm
Inc., 3550 General Atomics Ct., San Diego, California 92121, USA. Correspondence should be addressed to E.E.B. (emmbaetge@cytheraco.com)
Received 24 August; accepted 3 October; published online 28 October 2005; doi:10.1038/nbt1163
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Figure 1 Effect of serum concentration on hES cell–derived definitive endoderm production. (a) As determined by real-time quantitative PCR (Q-PCR),
SOX17, GSC and FOXA2, genes expressed in definitive endoderm, and MIXL1 are highly expressed after 5 d of hES cell differentiation only when high
dose activin A is provided. Furthermore, activin A is most effective when serum supplementation is lowest (>3 separate experiments). Numbers indicate
percent supplementation with FBS. NF indicates no activin A treatment whereas A100 indicates exposure to 100 ng/ml activin A. Y-axis indicates relative
gene expression normalized to the first sample in the data set (see methods). (b) Genes not expressed in definitive endoderm exhibit low level expression
under those conditions that robustly generate definitive endoderm. (c) SOX17 immunoreactive cells are most numerous under high dose activin A /low FBS.
SOX17+ cells are rare and occur only in isolated patches when no exogenous activin A is provided. Scale bar, 100 µm.
such as SOX17, GSC and FOXA2 (Fig. 1a)13–15,18,19, in addition to the
primitive streak marker MIXL1 (refs. 20,21). As these four genes are also
expressed in primitive/parietal/visceral endoderm, it is essential to monitor differentiation to this extra-embryonic lineage. Notably, expression of
SOX7, which is expressed in mouse primitive, parietal and visceral endoderm, but not in definitive endoderm18, was upregulated in the absence,
but not in the presence, of activin A (Fig. 1b). This indicates that the
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expression of SOX17, GSC and FOXA2 was not a result of differentiation
to primitive endoderm. Moreover, brachyury and MEOX1 expression was
low, showing that little mesoderm was generated22–24. SOX1 and ZIC1
expression was also low, demonstrating minimal ectoderm differentiation25–27 (Fig. 1b).
The relative proportion of hES cell–derived definitive endoderm produced under different fetal bovine serum (FBS) concentrations in the
presence or absence of activin A was shown at
the single-cell level by immunolocalization of
SOX17 (Fig. 1c; Supplementary Fig. 1 online).
The proportion of SOX17-positive cells was
greater than 80% after 5 d of differentiation
in 100 ng/ml activin A in 0.5% FBS, with no
detectable immunoreactivity for alpha-fetoprotein (AFP) or thrombomodulin (THBD),
markers of mouse and human visceral endoderm and mouse parietal endoderm/trophectoderm, respectively (data not shown).
Figure 2 The temporal dynamics of gene
expression indicates transition through a primitive
streak–like intermediate before expression of
definitive endoderm genes in activin A but not
BMP4/SU5402-treated cultures. (a) Treatment
of hES cells with activin A rapidly upregulates
expression of genes associated with proximal
epiblast (NODAL and FGF8) at 6 h and this is
followed by the peak expression of primitive
streak–expressed genes (WNT3 and brachyury) at
36 h. Expression of LHX1 also peaks at 36 h but
is subsequently maintained above hES cell levels
at 48 h and beyond. Expression of GSC, CER
and SOX17 is maximal at 96 h. (b–c) In contrast
to activin A treatment, BMP4/SU5402 induces
expression of genes characteristic of primitive
endoderm and trophectoderm lineages. A, 100
ng/ml activin A. B/SU, 100 ng/ml BMP4 and 5
µM SU5402.
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Transition through a primitive streak intermediate
Next we compared the dynamics of gene expression during differentiation of hES cells to definitive endoderm and extra-embryonic endoderm.
Application of BMP4 to hES cells results in differentiation to trophectoderm28, and endogenous BMP signaling in hES cell cultures is necessary
for differentiation to extra-embryonic endoderm29. In addition, blocking FGF signaling diminishes differentiation toward the mesoderm and
endoderm lineages (data not shown), consistent with genetic studies in
mice30–32. We found that exposure of hES cells to BMP4 in the presence
of the FGFR1 inhibitor, SU5402, results in differentiation primarily to
primitive endoderm and trophectoderm lineages (Fig. 2).
We examined the gene expression dynamics in either activin A or
BMP4/SU5402 cell-culture environments at short time increments
(Fig. 2a–c). Specifically, hES cells were cultured in the absence of FBS
for 24 h, in 0.2% FBS for the second 24 h and in 2% FBS for days 3
and 4, with continuous exposure to either 100 ng/ml of activin A to
induce definitive endoderm differentiation or to 100 ng/ml of BMP4
and 5 µM SU5402 to induce primitive endoderm and trophectoderm.
In cultures treated with activin A, NODAL expression was maximally
upregulated, and FGF8 was highly upregulated over hES cell levels,
after 6 h of treatment. This pattern of induction was not seen in
BMP4/SU5402-treated cultures.
Nodal and Fgf8 expression are some of the first indicators of posterior
pattern formation in the epiblast of mice. Their rapid upregulation in
differentiating hES cells suggests that the cells are initiating the transition toward a primitive streak–like cell population1. In the presence of
activin A, induction of the primitive streak markers WNT3, brachyury,
LHX1 and GSC5,19,22,33,34 also began at 6 h, and expression continued
to increase until 36 h. In contrast, little-to-no increased expression
was observed in the BMP4/SU5402-treated
cultures. With activin A, the genes brachyury
(Fig. 2a) and FGF4 (data not shown), which
a
are not expressed in mouse definitive endoderm22,35,36, were downregulated extremely
rapidly to at-or-below hES cell levels by 96
h. GSC, CER and SOX17 attained maximal
expression at 96 h, consistent with differentiation to a definitive endoderm fate, in
the cultures treated with activin A but not
with BMP4/SU540218,19,37–40. This temporal sequence of gene expression is similar to
that which occurs in the course of definitive
endoderm differentiation during vertebrate
Figure 3 Kinetics of mRNA and protein
expression determined by real-time Q-PCR,
western blotting and immunocytochemistry.
(a) Q-PCR analyses profile the dynamic expression
of several key genes during a 5 day definitive
endoderm differentiation in the presence of low
FBS/100 ng/ml of activin A. (b) The detection
of protein levels by western analyses in these
same cultures. The position and size (in kDa) of
molecular weight standards are indicated on the
right side of the panel. The predicted molecular
weights (in kDa) are as follows: OCT4, 39;
brachyury (BRACH), 47; SOX17, 44; FOXA2, 48;
E-cadherin, 80; N-cadherin, 82; GAPDH, 36.
(c) Immunofluorescent labeling of differentiating
cultures demonstrate coexpression of SOX17 with
brachyury and FOXA2. BRACH, brachyury, E-CAD,
E-cadherin, N-CAD, N-cadherin. Scale bar (top
left panel), 100 µm.
gastrulation and suggests that the hES cells may transition through
similar intermediates during this differentiation process.
Notably, there was a lack of expression of the mesoderm markers
MEOX1, FOXF1, KDR (also known as FLK1), BMP4 and CXCL12
(also known as SDF1) under high activin A and low FBS conditions16,23,24,41–45 (Supplementary Fig. 2 online). In contrast, removal
of activin A at 24 h (simulation of lower NODAL signaling) resulted
in much weaker induction of definitive endoderm gene expression
and a gain of mesoderm gene expression by day 4 of differentiation
(Supplementary Fig. 2 online). Finally, the robust upregulation of
either SOX7 and CDX1, or CDX2 and HCG, solely in the BMP4/
SU5402-treated cultures suggests that hES cells have the potential to
differentiate to primitive endoderm and trophectoderm, respectively,
under appropriate conditions46–49 (Fig. 2b and 2c). However, differentiation to these other lineages is minimal in activin A–treated
cultures. In addition, cell morphologies were noticeably different in
the activin A–treated cultures compared with the BMP4/SU5402treated cultures by 24–48 h of differentiation; by 96 h the difference
was considerable (Supplementary Fig. 3 online).
To corroborate real-time quantitative PCR analyses, we ascertained
protein expression levels and immunolocalization for various markers in activin A–treated cultures (Figs. 3 and 4). Western blot analyses
(Fig. 3b) showed that when the peak of brachyury protein is observed
(at 2 d of differentiation), brachyury mRNA expression (Fig. 3a) has
already decreased substantially from its peak a day earlier. Further illustrating this highly dynamic expression, brachyury protein was no longer
detectable at day 3. Figure 3c similarly shows a rapid loss of brachyury immunoreactivity between 48 h and 60 h of differentiation. At the
peak of brachyury protein expression, SOX17 protein just begins to be
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detectable (at 2 d of differentiation) and appears to peak around day 3
or 4, when brachyury is no longer detected (Fig. 3b).
To trace the origin of the SOX17-expressing cells, we characterized
SOX17 immunoreactivity over time. Figure 3c shows that the majority of SOX17+ cells at 36 h and 48 h of differentiation also expressed
brachyury. Specifically, of a total of 1,001 SOX17+ nuclei counted at
36 h, 719 were also brachyury immunoreactive (72%), whereas at 48
h, 956 SOX17+/brachyury+ were counted among 1,423 total SOX17+
nuclei (67%). The observation that SOX17 expression is initiated in
brachyury+ precursors further strengthens the conclusion that the
SOX17+ cells are definitive endoderm rather than primitive endoderm, because brachyury expression has not been identified in the
primitive endoderm lineage22.
To further explore the extent of definitive endoderm differentiation in
our cultures, we examined the expression of FOXA2, which, like SOX17,
is expressed in definitive endoderm13–15. FOXA2 shows a similar RNA
and protein expression profile as SOX17 in activin A–treated cultures
(Fig. 3a,b). Moreover, at 72 h of differentiation, the definitive endoderm
cells were SOX17/FOXA2 double positive and already represented ~60–
70% of all cells in the culture (Fig. 3c). Although FOXA2 is also expressed
in axial mesoderm, the coexpression of SOX17 and FOXA2 is indicative
of differentiation to definitive endoderm and not to mesoderm.
hES cell EMT
During vertebrate gastrulation, epiblast cells undergoing EMT at the
primitive streak alter expression of E-cadherin protein on the cell surface3. The combined actions of FGF and TGFβ signaling induce the
zinc-finger transcription factor SNAI1, which is a direct transcriptional
repressor of E-cadherin50. We observed that SNAI1 mRNA levels peaked
at 24–36 h, similarly to the primitive streak markers WNT3, brachyury
and FGF4 (Fig. 2 and data not shown). It was therefore of interest to
examine E-cadherin expression in relation to brachyury expression.
Undifferentiated hES cells did not express brachyury, but brachyury
protein was first detected at the periphery of most colonies by 12 h
after addition of activin A, rapidly spreading to the colonies’ interior
by 36–48 h (Fig. 4a). E-cadherin mRNA and protein levels decreased
4
Figure 4 Immunocytochemical analyses of
differentiating hES cells are consistent with an
EMT. (a) Brachyury immunoreactivity at 12 h is
visible in a narrow ring of cells at the periphery
of colonies, which subsequently widens toward
the center of the colonies. Co-staining for (b)
Brachyury and E-cadherin and (c) N-cadherin,
brachyury and SOX17. BRACH, brachyury; E-CAD,
E-cadherin; N-CAD, N-cadherin. Scale bars, 200
µm (a), 100 µm (b) and 200 µm (c).
significantly by 48 h of activin A treatment
(Fig. 3). By 12–24 h there was a decrease in
cell-surface immunolocalization of E-cadherin specifically in brachyury-positive cells
at the periphery of colonies (Fig. 4b). After 24
h, as the number of brachyury-expressing cells
increased, the loss of cell-surface E-cadherin in
brachyury-positive cells was more pronounced,
and was nearly complete by 48 h (Fig. 4b).
Coincident with the loss of epithelial Ecadherin protein, cells undergoing EMT at the
primitive streak start to express mesenchymal
markers, including N-cadherin on the cell surface51,52. As additional evidence for an EMT
occurring in activin A–exposed cultures, an increase in mRNA and protein expression of N-cadherin, which first appears in brachyury-positive cells and which displays reciprocal kinetics to the disappearance of
E-cadherin, was observed (Figs. 3a,b and 4c and Supplementary Fig. 4
online). In addition, noticeable changes in cell morphology occurred
during this period of differentiation, consistent with the cells acquiring
a mesenchymal character (Supplementary Fig. 3 online).
CXCR4 expression permits isolation of definitive endoderm
With the protocol described above we routinely observe that up to
80% SOX17 positive cells can be obtained. This represents one of the
most efficient differentiation procedures reported for production of
a specific cell type from hES cells. However, to further characterize
hES cell–derived definitive endoderm it may be preferable to obtain
starting material of the highest purity. Thus, we sought a cell-surface
marker that would permit further enrichment of the definitive endoderm population.
In the gastrulating mouse embryo, the gene encoding the chemokine receptor Cxcr4 is expressed in the definitive endoderm and mesoderm but not in primitive endoderm/visceral endoderm16. We found
that in cultures exposed to activin A and lower levels of FBS there is
an increase in CXCR4 mRNA, which mirrors the increase in definitive
endoderm markers (Fig. 5a; compare to Fig. 1a). We also observed that
the number of CXCR4-positive cells, as determined by flow cytometry,
was below 5% in undifferentiated hES cell cultures and increased to as
much as 90% during the 5-day treatment with activin A (>5 separate
experiments) (Fig. 5b and Supplementary Fig. 5 online). After 5 d of
differentiation, the CXCR4-negative cell population showed higher levels of POU5F1 (also known as OCT4) expression than did undifferentiated hES cells, suggesting that most of the CXCR4-negative cells retain
hES cell or primitive ectoderm character (Fig. 5c). Notably, when the
CXCR4-positive cells were isolated after 5 d of activin A treatment by
fluorescence-activated cell sorting (FACS), they were enriched in expression of definitive endoderm markers and MIXL1 (Fig. 5d) and depleted
in expression of markers of primitive endoderm, mesoderm and ectoderm (Fig. 5e) relative to the presorted population. The presorted, high-
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Figure 5 Isolation of CXCR4-positive cells using FACS further enriches hES cell–derived definitive endoderm to near homogeneity. (a) Expression of CXCR4
displays a pattern analogous to that of definitive endoderm expressed genes (compare to Fig. 1a). Numbers indicate percent supplementation with FBS.
(b) The number of CXCR4 immunoreactive cells rapidly increases from <5% to as high as 90% during 5 d of hES cell differentiation in high activin A /low
FBS conditions. (c) After 5 d of differentiation, the CXCR4-negative population retains high levels of OCT4 expression. (d) After differentiation in high
activin A /low FBS, the expression of SOX17, GSC and FOXA2, genes expressed in definitive endoderm, and MIXL1 occurs primarily in the CXCR4-positive
cells. The level of increased expression over the presorted population (A100) indicates that near 100% purity of hES cell–derived definitive endoderm has
been achieved considering the already enriched composition of definitive endoderm in the presorted population (70% CXCR4-positive cells). (e) The high
dose activin A–treated cultures (A100) exhibit low expression of genes characteristic of mesoderm (brachyury and MOX1), primitive endoderm (SOX7) and
neurectoderm (ZIC1). These expression levels are further depleted in the CXCR4-positive fraction. NF, no activin A treatment. A10, 10 ng/ml activin A. A100,
100 ng/ml activin A. CX–, CXCR4 negative. CX+, CXCR4 positive.
dose activin A–treated cells are already very low in primitive endoderm,
mesoderm and ectoderm marker expression relative to cultures treated
with low-dose or no activin A (Fig. 5e). Thus, the further depletion is
significant and it suggests that CXCR4 sorting enriches definitive endoderm to near 100% purity. Although CXCR4 has been reported to be
expressed in mesoderm16, it does not appear to be expressed in the minor
population of mesoderm cells that may arise in our activin A–exposed
cultures, as we observed a decrease in expression of mesoderm markers
in isolated CXCR4-positive cells. Similar observations were made with
another hES cell line, BG01 (Supplementary Fig. 6 online).
its derivatives, visceral endoderm and parietal endoderm, is very similar
to that of definitive endoderm (Supplementary Table 1 online). Here
we have shown that hES cells grown as monolayers in the presence
of high concentrations of activin A and low concentrations of serum
for 4–5 d produce cultures highly enriched for definitive endoderm.
Differentiation to definitive endoderm, but not to other lineages, is supported by two lines of evidence.
hES cell–derived definitive endoderm further differentiates
in vivo.
To assess the developmental competency of hES cell–derived definitive
endoderm cells to further differentiate into endoderm derivatives, we
transplanted them under the kidney capsule of severe combined immunodeficient (SCID) mice. After 5 weeks, we examined the expression of
CDX2, villin and hepatocyte-specific antigen (HSA) in histological sections of grafts as compared with control tissues (intestine for CDX2 and
villin; liver for HSA). The grafts contained organized structures of cells
that were immunoreactive for CDX2, villin or HSA (Fig. 6). Although
these structures are not fully mature tissues, their presence does suggest
that the definitive endoderm cells are competent to partially progress
through an endodermal differentiation program.
DISCUSSION
The initial cell lineage choices available to a differentiating hES cell are
definitive endoderm, ectoderm, mesoderm, primitive endoderm and
trophectoderm28. Given this broad range of outcomes and the fact that
most available markers are expressed in multiple cell lineages, specific
combinations of expressed and nonexpressed genes must be evaluated
to convincingly demonstrate differentiation to any particular lineage.
In particular, the gene expression pattern of primitive endoderm and
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Figure 6 Endodermal differentiation of hES cell–derived definitive
endoderm grafts. Expression of either CDX2 and villin or HSA is shown
in histological sections of intestine and liver, respectively, as well as
histological sections of 5-week-old kidney capsule hES cell–derived
definitive endoderm grafts. Scale bar (top left panel), 100 µm.
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First, robust expression of genes expressed in definitive endoderm
is found in conjunction with low expression of genes expressed in the
primitive endoderm, mesoderm, ectoderm and trophectoderm lineages.
Because Sox17 is expressed in mouse visceral endoderm and parietal
endoderm as well as in definitive endoderm18, we have used lack of
SOX718 mRNA (found in primitive endoderm but not definitive endoderm) and lack of AFP (visceral endoderm) and THBD (parietal endoderm) immunoreactivity as well as relative gene expression (data not
shown) to define the absence of primitive endoderm and its derivatives53,54. Sox17 is not expressed in the other lineages (mesoderm, ectoderm, trophectoderm) of the early mouse embryo18; thus the cells that
express SOX17 mRNA and protein must be definitive endoderm. The
finding that 80% or more of the population is SOX17/FOXA2 positive
at 4–5 d further demonstrates that production of definitive endoderm
from hES cells is orchestrated by high-dose activin A in the context of low
serum supplementation. Few mesoderm cells are produced, as shown
by low expression of FOXF1, MEOX1, KDR (FLK1), BMP4 and CXCL12
(SDF1) (Supplementary Fig. 2 online); little neuroectoderm is made as
evidenced by the absence of SOX1 and ZIC1 expression (Fig. 5e); and
little trophectoderm, as CDX2, THBD and HCG remain low (Fig. 2c and
data not shown). As an important control for our assay system, all these
negative markers of definitive endoderm are highly upregulated upon
either activin A removal at 24 h (Supplementary Fig. 2 online) or upon
BMP4/SU5402 (Fig. 2) treatment of hES cells. These trends of enriched
definitive endoderm gene expression and depleted expression of genes
expressed in other lineages are even more pronounced in highly purified
definitive endoderm cells isolated by CXCR4 expression using FACS.
Second, the findings that highly enriched definitive endoderm arises
from cultures that are predominantly brachyury positive and that the
majority of early SOX17-positive cells costain for brachyury reinforce
the conclusion that definitive endoderm, and not primitive endoderm,
is generated. These transient brachyury-expressing cells may represent
mesendodermal progenitors that subsequently acquire a definitive endoderm fate as judged by the rapid-upregulation of SOX17 in cells with low/
absent expression of brachyury. Moreover, activin A–exposed hES cells
appear to undergo an EMT, analogous to that occurring in the primitive
streak of mice during the generation of mesendoderm cells3,55.
The intermediate stages in the differentiation of hES cells to definitive
endoderm—in particular, the temporal dynamics of gene expression
and the localization of specific proteins—resembles events in mouse
gastrulation. Rapid upregulation of NODAL and FGF8 expression at 6
h of differentiation may signify the initial transition towards primitive
streak–like cells; this is followed by upregulation of classical primitive
streak markers such as brachyury, FGF4 and WNT3, which occurs maximally at 24–36 h1. Subsequently, the primitive streak markers brachyury
and FGF4 exhibit rapid downregulation concurrent with the upregulation of definitive endoderm genes not expressed in the primitive streak,
such as SOX17 and CER. Continued expression of MIXL1 and LHX1 at
4–5 d in activin A–exposed hES cells was not predicted, as in mice these
genes are expressed in the primitive streak and mesoderm derivatives
but not in the definitive endoderm. We do, however, observe that MIXL1
and LHX1 expression at 4 d are lower than at 24–36 h. Given the large
proportion of cells that are SOX17/FOXA2 immunoreactive at 4–5 d,
the cells that express MIXL1 and LHX1 are either rare primitive streak
and mesoderm cells or represent a difference in regulation between the
mouse and human genes.
Recently described protocols12,56 to differentiate mouse ES cells to
definitive endoderm using activin A differ from our approach in the culture methods used and in the markers of definitive endoderm formation.
In our hands, these differentiation conditions do not produce any definitive endoderm from hES cells (data not shown). However, as mouse and
6
human ES cells require different culture conditions to maintain them
in an undifferentiated state57, the requirements for differentiation to
definitive endoderm may be appreciably different as well.
Interestingly, hES cells engineered to constitutively express the TGFβ
ligand Nodal fail to differentiate into definitive endoderm or mesoderm
either in monolayer or embryoid body suspension culture58. However,
these experiments were performed under conditions quite different from
those described here. It appears that under conditions optimized for
growth of undifferentiated hES cells, TGFβ ligands promote maintenance of pluripotency58–61.
Finally, to assess whether our method for generating definitive endoderm is universally applicable, we evaluated seven additional hES cell
lines. Each of the seven lines exhibited gene expression dynamics similar
to that of CyT25 in Figure 2 (Supplementary Fig. 7 online). The H7
and H9 hES cell lines were maintained on Matrigel in the absence of
fibroblast feeders, further demonstrating that this differentiation procedure will be broadly applicable. Interestingly, the different hES cell lines
produce definitive endoderm with varying efficiencies, perhaps because
of variations in the propensity of the primitive streak/mesendodermlike state to convert to definitive endoderm rather than mesoderm. For
instance, during differentiation of the H7 and the three BG hES cell
lines, brachyury gene expression, after its initial peak, did not rapidly
drop to levels near those of undifferentiated hES cells (Supplementary
Fig. 7 online).
In summary, we have described an approach to produce highly
enriched cultures of definitive endoderm from hES cells. This work suggests that differentiation of hES cells may serve as a model of early vertebrate development (Supplementary Fig. 8 online). The production of
hES cell–derived definitive endoderm is also a critical step in generating
scientifically and therapeutically useful cells of the definitive endoderm
lineage, such as hepatocytes and pancreatic endocrine cells.
METHODS
Cell culture. Undifferentiated hES cells were maintained on mouse embryo fibroblast feeder layers (Specialty Media) in DMEM/F12 (Mediatech) supplemented
with 20% KnockOut serum replacement (Gibco), 1 mM nonessential amino acids
(Gibco), Glutamax (Gibco), penicillin/streptomycin (Gibco), 0.55 mM 2-mercaptoethanol (Gibco) and 4 ng/ml recombinant human FGF2 (R&D Systems).
On occasion, activin A is added to hES cell growth medium at 10 ng/ml to help
maintain undifferentiated growth. Cultures were manually passaged at 1:4–1:10
split ratio every 5–7 d. Differentiation was carried out in RPMI (Mediatech)
supplemented with Glutamax, penicillin/streptomycin and varying concentrations of defined FBS (HyClone). Before initiating differentiation, hES cells were
given a brief wash in PBS+/+ (Gibco). In most differentiation experiments FBS
concentrations were 0% for the first 24 h, 0.2% for the second 24 h, and 2.0% for
subsequent days of differentiation. Recombinant human activin A and BMP4
were purchased from R&D Systems. SU5402 was a kind gift of A.Terskikh.
Production of SOX17 antibody. The SOX17 antibody was produced by genetic
immunization in rats by and according to procedures developed at GENOVAC.
A portion of the human SOX17 cDNA corresponding to amino acids 172–414
in the C-terminal end of SOX17 was used for immunization.
Western blot analyses. Cells were harvested in lysis solution (50 mM Tris [pH
8.0], 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% deoxycholate) supplemented
with a cocktail of protease inhibitors (Roche). Proteins were separated on Bis-Tris
polyacrylamide gels (NuPage 4–12%, Invitrogen), transferred by electroblotting
onto PDVF membranes (Bio-Rad) and detected through horseradish-peroxidase
conjugated secondary antibodies (Jackson ImmunoResearch Laboratories) and
chemiluminescent (ECL Plus, Amersham) exposure of BioMax film (Kodak). The
following dilutions were used for primary antibodies: rat anti-SOX17 (Genovac),
1:1,000; goat anti-brachyury (R&D systems, AF2085), 1:1,000; goat anti-Oct4
(Santa Cruz, sc-9081), 1:200; goat anti-FOXA2 (R&D systems, AF2400), 1:1,000;
mouse anti-E-cadherin (Zymed, 13-1700), 1:1,000; mouse anti-N-cadherin
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(BD, 610921), 1:2,500; mouse anti-GAPDH (Chemicon, MAB374), 1:500; mouse
anti-EGFP (Clontech, no. 632381), 1:1,000.
Immunostaining. Cultures were fixed for 15 min at 18–25 °C in 4% wt/vol paraformaldehyde in PBS, washed several times in Tris-buffered saline (TBS) and
blocked for 30 min in TBST (TBS/0.25% wt/vol Triton X-100 (Sigma)) containing 3% normal donkey serum (nds, Jackson ImmunoResearch Laboratories).
Primary and secondary antibodies were diluted in TBST/3% nds and incubated
for 24 h at 4 °C or 2 h at room temperature. The following antibodies and dilutions were used: rat anti-SOX17, 1:500; goat anti-brachyury, 1:50 (R&D Systems);
mouse anti E-cadherin, 1:200 (Zymed); mouse anti N-cadherin, 1:300 (BD),
goat anti-FOXA2, 1:50 (R&D Systems); donkey anti-rat-Cy3,1:400 (Jackson
ImmunoResearch Laboratories, JIRL); donkey anti-mouse-FITC, 1:100 (JIRL),
donkey anti-goat-Cy2, 1:200 (JIRL); donkey anti-goat-Cy3, 1:400 (JIRL);
Alexa-488 and Alexa-555 conjugated donkey antibodies against mouse and goat
(Molecular Probes), 1:500. Immunostaining of paraffin-embedded graft sections were performed with monoclonal antibodies to hepatocyte-specific antigen
(HSA, Cell Marque, OCH1E5, 1:50), villin (Neomarkers, MS-1499-R7, 1:50) and
CDX2 (Abcam, ab15258, 1:25) and the mouse-to-mouse (Chemicon, #2700-S)
horseradish peroxidase kit.
Real-time quantitative PCR. Total RNA was isolated from duplicate or triplicate samples and 100-500 ng used for reverse transcription with iScript cDNA
synthesis kit (Biorad). PCR reactions were run in duplicate using 1/40th of the
cDNA per reaction and 400 nM forward and reverse primers with Quantitect
SYBR Green master mix (Qiagen). Alternatively, Taqman gene expression assays
were used according to the manufacturer’s instructions (Applied Biosystems).
Real-time PCR was performed using the Rotor Gene 3000 (Corbett Research).
Relative quantification was performed against a standard curve and quantified
values were normalized against the input determined by two housekeeping genes
(CYCG and GUSB or TBP). After normalization, the samples were plotted relative to the first sample in the data set and the standard deviation of the 4 or 6
gene expression measurements is reported. Primer sequences are reported in
Supplementary Table 1 online.
FACS. Cells were partially dissociated using 0.05% trypsin/EDTA (Invitrogen)
for ~1 min followed by removal of trypsin and further dissociation in PBS with
2% human serum (buffer). Cells were pelleted and resuspended in human serum
to block nonspecific antibody binding. Cells were labeled with CXCR4-PE (R&D
Systems) at 10 µl per 2.5 × 105 cells for 45 min on ice. Cells were washed twice in
buffer and resuspended in buffer at 5 × 106/ml. Cells were analyzed and isolated
using a FACS Vantage (BD Bioscience) by the staff at the flow cytometry core
(TSRI). Cells were collected into RLT lysis buffer and total RNA isolated using
RNeasy according to the manufacturer’s instructions (Qiagen).
Kidney capsule implantation. hES cell–derived definitive endoderm culture
plates were washed with 2% FBS in RPMI medium and scored with a needle
into ~2- to 4-mm squares. Cell clumps were lifted with a cell scraper, collected by
centrifugation and resuspended in 2% FBS in RPMI. About 25 µl (~106 cells) of
cell clumps were implanted into the left kidney of ketamine/xylazine anesthetized
SCID mice (housed at the B. Braun Biological Test Center, Irvine CA).
Note: Supplementary information is available on the Nature Biotechnology website.
ACKNOWLEDGMENTS
We wish to acknowledge Melissa Carpenter for culture and differentiation of H7
and H9 hES cells, Gillian Beattie for culture and differentiation of HUES7 hES cells
and Bobbie Daughters and Jie Zheng for kidney capsule implantation and histology,
respectively. Special thanks to Melissa Carpenter, Anne Bang, Matthias Hebrok,
Didier Stainier and Jim Wells for helpful advice and critical review of the manuscript.
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing financial interests.
Published online at http://www.nature.com/naturebiotechnology/
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions/
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