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Efficient differentiation of human embryonic stem cells to definitive endodermKevin 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 respira-tory and digestive tracts and to the thyroid, thymus, lungs, liver and pan-creas. 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 epi-thelial-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 primi-tive 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 sig-naling specifying mesoderm9,10. Although Nodal is an attractive can-didate 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

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 produc-tion 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 indi-cate that the temporal sequence of gene expression characteristic of hES cell differentiation to definitive endoderm is similar to the transi-tions 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).

RESULTSSerum concentration affects definitive endoderm productionBecause Nodal/activin induces endoderm in vertebrates17, we tested the effects of activin A on human ES cells. Lowering the serum concentra-tion in the presence of 100 ng/ml activin A for 5 d resulted in maximally elevated expression of genes expressed in vertebrate definitive endoderm

1CyThera 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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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 moni-tor differentiation to this extra-embryonic lineage. Notably, expression of SOX7, which is expressed in mouse primitive, parietal and visceral endo-derm, but not in definitive endoderm18, was upregulated in the absence, but not in the presence, of activin A (Fig. 1b). This indicates that the

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 differentia-tion25–27 (Fig. 1b).

The relative proportion of hES cell–derived definitive endoderm pro-duced 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-feto-protein (AFP) or thrombomodulin (THBD), markers of mouse and human visceral endo-derm and mouse parietal endoderm/trophec-toderm, respectively (data not shown).

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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.

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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 intermediateNext we compared the dynamics of gene expression during differentia-tion of hES cells to definitive endoderm and extra-embryonic endoderm. Application of BMP4 to hES cells results in differentiation to trophecto-derm28, and endogenous BMP signaling in hES cell cultures is necessary for differentiation to extra-embryonic endoderm29. In addition, block-ing 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 transi-tion 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 are not expressed in mouse definitive endo-derm22,35,36, 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 differ-entiation to a definitive endoderm fate, in the cultures treated with activin A but not with BMP4/SU540218,19,37–40. This tempo-ral sequence of gene expression is similar to that which occurs in the course of definitive endoderm differentiation during vertebrate

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 condi-tions16,23,24,41–45 (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, dif-ferentiation 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/SU5402-treated 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 mark-ers 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 illus-trating this highly dynamic expression, brachyury protein was no longer detectable at day 3. Figure 3c similarly shows a rapid loss of brachy-ury 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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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.

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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 major-ity 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 endo-derm, 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 EMTDuring vertebrate gastrulation, epiblast cells undergoing EMT at the primitive streak alter expression of E-cadherin protein on the cell sur-face3. 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

significantly by 48 h of activin A treatment (Fig. 3). By 12–24 h there was a decrease in cell-surface immunolocalization of E-cad-herin 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 E-cadherin protein, cells undergoing EMT at the primitive streak start to express mesenchymal markers, including N-cadherin on the cell sur-face51,52. As additional evidence for an EMT

occurring in activin A–exposed cultures, an increase in mRNA and pro-tein expression of N-cadherin, which first appears in brachyury-posi-tive 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 endodermWith 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 endo-derm population.

In the gastrulating mouse embryo, the gene encoding the chemo-kine receptor Cxcr4 is expressed in the definitive endoderm and meso-derm 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 lev-els of POU5F1 (also known as OCT4) expression than did undifferenti-ated 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 expres-sion of definitive endoderm markers and MIXL1 (Fig. 5d) and depleted in expression of markers of primitive endoderm, mesoderm and ecto-derm (Fig. 5e) relative to the presorted population. The presorted, high-

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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).

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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 endo-derm 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).

hES cell–derived definitive endoderm further differentiatesin 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 immu-nodeficient (SCID) mice. After 5 weeks, we examined the expression of CDX2, villin and hepatocyte-specific antigen (HSA) in histological sec-tions 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.

DISCUSSIONThe 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

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 sup-ported by two lines of evidence.

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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.

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 endo-derm) and lack of AFP (visceral endoderm) and THBD (parietal endo-derm) immunoreactivity as well as relative gene expression (data not shown) to define the absence of primitive endoderm and its deriva-tives53,54. Sox17 is not expressed in the other lineages (mesoderm, ecto-derm, 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 endo-derm 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 maxi-mally at 24–36 h1. Subsequently, the primitive streak markers brachyury and FGF4 exhibit rapid downregulation concurrent with the upregula-tion 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 cul-ture methods used and in the markers of definitive endoderm formation. In our hands, these differentiation conditions do not produce any defini-tive endoderm from hES cells (data not shown). However, as mouse and

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 mainte-nance of pluripotency58–61.

Finally, to assess whether our method for generating definitive endo-derm 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 proce-dure 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/mesendoderm-like 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 sug-gests that differentiation of hES cells may serve as a model of early ver-tebrate 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.

METHODSCell culture. Undifferentiated hES cells were maintained on mouse embryo fibro-blast 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-mer-captoethanol (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 concentra-tions 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 para-formaldehyde 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)) contain-ing 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 dilu-tions 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 sec-tions 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 tripli-cate 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 rela-tive 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.

ACKNOWLEDGMENTSWe 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 STATEMENTThe 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/

1. Lu, C.C., Brennan, J. & Robertson, E.J. From fertilization to gastrulation: axis formation in the mouse embryo. Curr. Opin. Genet. Dev. 11, 384–392 (2001).

2. Robb, L. & Tam, P.P. Gastrula organiser and embryonic patterning in the mouse. Semin. Cell Dev. Biol. 15, 543–554 (2004).

3. Shook, D. & Keller, R. Mechanisms, mechanics and function of epithelial-mesenchymal transitions in early development. Mech. Dev. 120, 1351–1383 (2003).

4. Haegel, H. et al. Lack of beta-catenin affects mouse development at gastrulation. Development 121, 3529–3537 (1995).

5. Liu, P. et al. Requirement for Wnt3 in vertebrate axis formation. Nat. Genet. 22, 361–365 (1999).

6. Kelly, O.G., Pinson, K.I. & Skarnes, W.C. The Wnt co-receptors Lrp5 and Lrp6 are essential for gastrulation in mice. Development 131, 2803–2815 (2004).

7. Conlon, F.L. et al. A primary requirement for nodal in the formation and maintenance of the primitive streak in the mouse. Development 120, 1919–1928 (1994).

8. Brennan, J. et al. Nodal signalling in the epiblast patterns the early mouse embryo. Nature 411, 965–969 (2001).

9. Lowe, L.A., Yamada, S. & Kuehn, M.R. Genetic dissection of nodal function in patterning the mouse embryo. Development 128, 1831–1843 (2001).

10. Vincent, S.D., Dunn, N.R., Hayashi, S., Norris, D.P. & Robertson, E.J. Cell fate deci-sions within the mouse organizer are governed by graded Nodal signals. Genes Dev. 17, 1646–1662 (2003).

11. de Caestecker, M. The transforming growth factor-beta superfamily of receptors. Cytokine Growth Factor Rev. 15, 1–11 (2004).

12. Kubo, A. et al. Development of definitive endoderm from embryonic stem cells in culture. Development 131, 1651–1662 (2004).

13. Sasaki, H. & Hogan, B.L. Differential expression of multiple fork head related genes during gastrulation and axial pattern formation in the mouse embryo. Development 118, 47–59 (1993).

14. Ang, S.L. et al. The formation and maintenance of the definitive endoderm lineage in the mouse: involvement of HNF3/forkhead proteins. Development 119, 1301–1315 (1993).

15. Monaghan, A.P., Kaestner, K.H., Grau, E. & Schutz, G. Postimplantation expression patterns indicate a role for the mouse forkhead/HNF-3 alpha, beta and gamma genes in determination of the definitive endoderm, chordamesoderm and neuroectoderm. Development 119, 567–578 (1993).

16. McGrath, K.E., Koniski, A.D., Maltby, K.M., McGann, J.K. & Palis, J. Embryonic expres-sion and function of the chemokine SDF-1 and its receptor, CXCR4. Dev. Biol. 213, 442–456 (1999).

17. Stainier, D.Y. A glimpse into the molecular entrails of endoderm formation. Genes Dev. 16, 893–907 (2002).

18. Kanai-Azuma, M. et al. Depletion of definitive gut endoderm in Sox17-null mutant mice. Development 129, 2367–2379 (2002).

19. Blum, M. et al. Gastrulation in the mouse: the role of the homeobox gene goosecoid. Cell 69, 1097–1106 (1992).

20. Hart, A.H. et al. Mixl1 is required for axial mesendoderm morphogenesis and patterning in the murine embryo. Development 129, 3597–3608 (2002).

21. Pearce, J.J. & Evans, M.J. Mml, a mouse Mix-like gene expressed in the primitive streak. Mech. Dev. 87, 189–192 (1999).

22. Wilkinson, D.G., Bhatt, S. & Herrmann, B.G. Expression pattern of the mouse T gene and its role in mesoderm formation. Nature 343, 657–659 (1990).

23. Candia, A.F. et al. Mox-1 and Mox-2 define a novel homeobox gene subfamily and are differentially expressed during early mesodermal patterning in mouse embryos. Development 116, 1123–1136 (1992).

24. Candia, A.F. & Wright, C.V. Differential localization of Mox-1 and Mox-2 proteins indi-cates distinct roles during development. Int. J. Dev. Biol. 40, 1179–1184 (1996).

25. Pevny, L.H., Sockanathan, S., Placzek, M. & Lovell-Badge, R. A role for SOX1 in neural determination. Development 125, 1967–1978 (1998).

26. Nagai, T. et al. The expression of the mouse Zic1, Zic2, and Zic3 gene suggests an essen-tial role for Zic genes in body pattern formation. Dev. Biol. 182, 299–313 (1997).

27. Elms, P. et al. Overlapping and distinct expression domains of Zic2 and Zic3 during mouse gastrulation. Gene Expr. Patterns 4, 505–511 (2004).

28. Xu, R.H. et al. BMP4 initiates human embryonic stem cell differentiation to trophoblast. Nat. Biotechnol. 20, 1261–1264 (2002).

29. Pera, M.F. et al. Regulation of human embryonic stem cell differentiation by BMP-2 and its antagonist noggin. J. Cell Sci. 117, 1269–1280 (2004).

30. Yamaguchi, T.P., Harpal, K., Henkemeyer, M. & Rossant, J. fgfr-1 is required for embryonic growth and mesodermal patterning during mouse gastrulation. Genes Dev. 8, 3032–3044 (1994).

31. Deng, C.X. et al. Murine FGFR-1 is required for early postimplantation growth and axial organization. Genes Dev. 8, 3045–3057 (1994).

32. Sun, X., Meyers, E.N., Lewandoski, M. & Martin, G.R. Targeted disruption of Fgf8 causes failure of cell migration in the gastrulating mouse embryo. Genes Dev. 13, 1834–1846 (1999).

33. Barnes, J.D., Crosby, J.L., Jones, C.M., Wright, C.V. & Hogan, B.L. Embryonic expression of Lim-1, the mouse homolog of Xenopus Xlim-1, suggests a role in lateral mesoderm differentiation and neurogenesis. Dev. Biol. 161, 168–178 (1994).

34. Shawlot, W. & Behringer, R.R. Requirement for Lim1 in head-organizer function. Nature 374, 425–430 (1995).

35. Niswander, L. & Martin, G.R. Fgf-4 expression during gastrulation, myogenesis, limb and tooth development in the mouse. Development 114, 755–768 (1992).

36. Rappolee, D.A., Basilico, C., Patel, Y. & Werb, Z. Expression and function of FGF-4 in peri-implantation development in mouse embryos. Development 120, 2259–2269 (1994).

37. Belo, J.A. et al. Cerberus-like is a secreted factor with neutralizing activity expressed in the anterior primitive endoderm of the mouse gastrula. Mech. Dev. 68, 45–57 (1997).

ART ICLES©

2005

Nat

ure

Pub

lishi

ng G

roup

ht

tp://

ww

w.n

atur

e.co

m/n

atur

ebio

tech

nolo

gy

8 ADVANCE ONLINE PUBLICATION NATURE BIOTECHNOLOGY

38. Biben, C. et al. Murine cerberus homologue mCer-1: a candidate anterior patterning molecule. Dev. Biol. 194, 135–151 (1998).

39. Shawlot, W., Deng, J.M. & Behringer, R.R. Expression of the mouse cerberus-related gene, Cerr1, suggests a role in anterior neural induction and somitogenesis. Proc. Natl. Acad. Sci. USA 95, 6198–6203 (1998).

40. Pearce, J.J., Penny, G. & Rossant, J. A mouse cerberus/Dan-related gene family. Dev. Biol. 209, 98–110 (1999).

41. Mahlapuu, M., Ormestad, M., Enerback, S. & Carlsson, P. The forkhead transcription factor Foxf1 is required for differentiation of extra-embryonic and lateral plate meso-derm. Development 128, 155–166 (2001).

42. Ormestad, M., Astorga, J. & Carlsson, P. Differences in the embryonic expression pat-terns of mouse Foxf1 and -2 match their distinct mutant phenotypes. Dev. Dyn. 229, 328–333 (2004).

43. Shalaby, F. et al. Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice. Nature 376, 62–66 (1995).

44. Winnier, G., Blessing, M., Labosky, P.A. & Hogan, B.L. Bone morphogenetic protein-4 is required for mesoderm formation and patterning in the mouse. Genes Dev. 9, 2105–2116 (1995).

45. Lawson, K.A. et al. Bmp4 is required for the generation of primordial germ cells in the mouse embryo. Genes Dev. 13, 424–436 (1999).

46. Meyer, B.I. & Gruss, P. Mouse Cdx-1 expression during gastrulation. Development 117, 191–203 (1993).

47. McGrath, K.E. & Palis, J. Expression of homeobox genes, including an insulin promot-ing factor, in the murine yolk sac at the time of hematopoietic initiation. Mol. Reprod. Dev. 48, 145–153 (1997).

48. Strumpf, D. et al. Cdx2 is required for correct cell fate specification and differentiation of trophectoderm in the mouse blastocyst. Development 132, 2093–2102 (2005).

49. Lacroix, M.C., Guibourdenche, J., Frendo, J.L., Pidoux, G. & Evain-Brion, D. Placental growth hormones. Endocrine 19, 73–79 (2002).

50. Batlle, E. et al. The transcription factor snail is a repressor of E-cadherin gene expres-sion in epithelial tumour cells. Nat. Cell Biol. 2, 84–89 (2000).

51. Hatta, K. & Takeichi, M. Expression of N-cadherin adhesion molecules associated with early morphogenetic events in chick development. Nature 320, 447–449 (1986).

52. Radice, G.L. et al. Developmental defects in mouse embryos lacking N-cadherin. Dev. Biol. 181, 64–78 (1997).

53. Dziadek, M.A. & Andrews, G.K. Tissue specificity of alpha-fetoprotein messenger RNA expression during mouse embryogenesis. EMBO J. 2, 549–554 (1983).

54. Weiler-Guettler, H., Aird, W.C., Rayburn, H., Husain, M. & Rosenberg, R.D. Developmentally regulated gene expression of thrombomodulin in postimplantation mouse embryos. Development 122, 2271–2281 (1996).

55. Ciruna, B.G., Schwartz, L., Harpal, K., Yamaguchi, T.P. & Rossant, J. Chimeric analysis of fibroblast growth factor receptor-1 (Fgfr1) function: a role for FGFR1 in morphogenetic movement through the primitive streak. Development 124, 2829–2841 (1997).

56. Tada, S. et al. Characterization of mesendoderm: a diverging point of the definitive endoderm and mesoderm in embryonic stem cell differentiation culture. Development 132, 4363–4374 (2005).

57. Wei, C.L. et al. Transcriptome profiling of human and murine ESCs identifies divergent paths required to maintain the stem cell state. Stem Cells 23, 166–185 (2005).

58. Vallier, L., Reynolds, D. & Pedersen, R.A. Nodal inhibits differentiation of human embryonic stem cells along the neuroectodermal default pathway. Dev. Biol. 275, 403–421 (2004).

59. Amit, M., Shariki, C., Margulets, V. & Itskovitz-Eldor, J. Feeder layer- and serum-free culture of human embryonic stem cells. Biol. Reprod. 70, 837–845 (2004).

60. Beattie, G.M. et al. Activin A maintains pluripotency of human embryonic stem cells in the absence of feeder layers. Stem Cells 23, 489–495 (2005).

61. James, D., Levine, A.J., Besser, D. & Hemmati-Brivanlou, A. TGFbeta/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells. Development 132, 1273–1282 (2005).

ART ICLES©

2005

Nat

ure

Pub

lishi

ng G

roup

ht

tp://

ww

w.n

atur

e.co

m/n

atur

ebio

tech

nolo

gy