The development of functional cell populations such hepatocytes and pancreatic beta cells from embryonic stem (ES) cells is dependent on the efficient induction of definitive endoderm early in the differentiation process. To monitor definitive endoderm formation in mouse ES cell differentiation cultures in a quantitative fashion, we generated a reporter cell line that expresses human CD25 from the Foxa3 locus and human CD4 from the Foxa2 locus. Induction of these reporter ES cells with high concentrations of Activin A (activin) led to the development of a CD25-Foxa3+CD4-Foxa2+ population within four to five days of culture. Isolation and characterization of this population revealed that it consists predominantly of definitive endoderm that is able to undergo hepatic specification under the appropriate conditions. To develop reagents that can be used for studies on endoderm development from un-manipulated ES cells, from induced pluripotent stem (iPS) cells, and from the mouse embryo we generated monoclonal antibodies against the CD25-Foxa3+CD4-Foxa2+ population. With this approach, we identified two antibodies that react specifically with endoderm from ES cell cultures as well as from the early embryo. The specificity of these antibodies enables one to quantitatively monitor endoderm development in ES cell differentiation cultures, to study endoderm formation in the embryo and to isolate pure populations of culture- or embryo-derived endodermal cells.
Embryonic stem cells; Monoclonal antibodies; Embryoid bodies; Differentiation antigens; Differentiation
Complex cross-talk between endoderm and the microenvironment is an absolute requirement to orchestrate hepatic specification and expansion. In the mouse, the septum transversum and cardiac mesoderm, through secreted BMPs and FGFs, respectively, instruct the adjacent ventral endoderm to become hepatic endoderm. Consecutively, endothelial cells promote expansion of the specified hepatic endoderm. Using a mouse reporter embryonic stem (ES) cell line in which hCD4 and hCD25 were targeted to the Foxa2 and Foxa3 loci, we reconstituted an in vitro culture system in which committed endoderm cells co-expressing hCD4-Foxa2 and hCD25-Foxa3 were isolated, and co-cultured with endothelial cells in the presence of BMP4 and bFGF. In this culture setting, we provide mechanistic evidence that endothelial cells function not only to promote hepatic endoderm expansion, but are also required at an earlier step for hepatic specification, at least in part through regulation of the Wnt and Notch pathways. Activation of Wnt and Notch by chemical or genetic approaches increases endoderm cell numbers but inhibits hepatic specification, and conversely, chemical inhibition of both pathways enhances hepatic specification and reduces proliferation. Using identical co-culture conditions, we defined a similar dependence of endoderm harvested from embryos on endothelial cells to support their growth and hepatic specification. Our findings (1) confirm a conserved role of Wnt repression for mouse hepatic specification, (2) uncover a novel role for Notch repression in the hepatic fate decision, and (3) demonstrate that repression of Wnt and Notch signaling in hepatic endoderm is controlled by the endothelial cell niche.
Endoderm; endothelial cells; mouse embryonic stem cells; mouse embryo; Wnt; Notch
Unrestricted somatic stem cells (USSC) derived from umbilical cord blood are an attractive alternative to human embryonic stem cells (hESC) for cellular therapy. USSC are capable of forming cells representative of all three germ line layers. The aim of this study was to determine the potential of USSC to form definitive endoderm following induction with Activin A, a protein known to specify definitive endoderm formation of hESC.
USSC were cultured for (1) three days with or without 100 ng/ml Activin A in either serum-free, low-serum or serum-containing media, (2) three days with or without 100 ng/ml Activin A in combination with 10 ng/ml FGF4 in pre-induction medium, or (3) four days with or without small molecules Induce Definitive Endoderm (IDE1, 100 nM; IDE2, 200 nM) in serum-free media. Formation of definitive endoderm was assessed using RT-PCR for gene markers of endoderm (Sox17, FOXA2 and TTF1) and lung epithelium (surfactant protein C; SPC) and cystic fibrosis transmembrane conductance regulator; CFTR). The differentiation capacity of Activin A treated USSC was also assessed.
Activin A or IDE1/2 induced formation of Sox17+ definitive endoderm from hESC but not from USSC. Activin A treated USSC retained their capacity to form cells of the ectoderm (nerve), mesoderm (bone) and endoderm (lung). Activin A in combination with FGF4 did not induce formation of Sox17+ definitive endoderm from USSC. USSC express both Activin A receptor subunits at the mRNA and protein level, indicating that these cells are capable of binding Activin A.
Stimulation of the Nodal signaling pathway with Activin A or IDE1/2 is insufficient to induce definitive endoderm formation from USSC, indicating that USSC differ in their stem cell potential from hESC.
Embryonic stem cells (ESCs) are derived from mammalian embryos during the transition from totipotency, when individual blastomeres can make all lineages, to pluripotency, when they are competent to make only embryonic lineages. ESCs maintained with inhibitors of MEK and GSK3 (2i) are thought to represent an embryonically restricted ground state. However, we observed heterogeneous expression of the extraembryonic endoderm marker Hex in 2i-cultured embryos, suggesting that 2i blocked development prior to epiblast commitment. Similarly, 2i ESC cultures were heterogeneous and contained a Hex-positive fraction primed to differentiate into trophoblast and extraembryonic endoderm. Single Hex-positive ESCs coexpressed epiblast and extraembryonic genes and contributed to all lineages in chimeras. The cytokine LIF, necessary for ESC self-renewal, supported the expansion of this population but did not directly support Nanog-positive epiblast-like ESCs. Thus, 2i and LIF support a totipotent state comparable to early embryonic cells that coexpress embryonic and extraembryonic determinants.
•Embryos cultured in 2i are blocked before extraembryonic lineage segregation•ESCs cultured in 2i express extraembryonic markers heterogeneously•Single 2i ESCs differentiate into both embryonic and extraembryonic lineages•LIF promotes proliferation of extraembryonically primed ESC populations
Embryonic stem cells (ESCs) are isolated from preimplantation embryos and can contribute to all tissues of the embryo, but not extraembryonic tissues (e.g., placenta). Therefore, they are considered pluripotent, not totipotent. Brickman and colleagues now show that single ESCs cultured in the presence of MEK and GSK3 inhibitors (2i) coexpress embryonic and extraembryonic markers and contribute to both embryonic and extraembryonic tissues (e.g., are totipotent). A role of the cytokine LIF in ESC self-renewal is to support this totipotent population.
The establishment of methods for directive differentiation from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) is important for regenerative medicine. Although Sry-related HMG box 17 (SOX17) overexpression in ESCs leads to differentiation of either extraembryonic or definitive endoderm cells, respectively, the mechanism of these distinct results remains unknown. Therefore, we utilized a transient adenovirus vector-mediated overexpression system to mimic the SOX17 expression pattern of embryogenesis. The number of alpha-fetoprotein-positive extraembryonic endoderm (ExEn) cells was increased by transient SOX17 transduction in human ESC- and iPSC-derived primitive endoderm cells. In contrast, the number of hematopoietically expressed homeobox (HEX)-positive definitive endoderm (DE) cells, which correspond to the anterior DE in vivo, was increased by transient adenovirus vector-mediated SOX17 expression in human ESC- and iPSC-derived mesendoderm cells. Moreover, hepatocyte-like cells were efficiently generated by sequential transduction of SOX17 and HEX. Our findings show that a stage-specific transduction of SOX17 in the primitive endoderm or mesendoderm promotes directive ExEn or DE differentiation by SOX17 transduction, respectively.
Nodal and Activin are morphogens of the TGFbeta superfamily of signaling molecules that direct differential cell fate decisions in a dose- and distance-dependent manner. During early embryonic development the Nodal/Activin pathway is responsible for the specification of mesoderm, endoderm, node, and mesendoderm. In contradiction to this drive towards cellular differentiation, the pathway also plays important roles in the maintenance of self-renewal and pluripotency in embryonic and epiblast stem cells. The molecular basis behind stem cell interpretation of Nodal/Activin signaling gradients and the undertaking of disparate cell fate decisions remains poorly understood. Here, we show that any perturbation of endogenous signaling levels in mouse embryonic stem cells leads to their exit from self-renewal towards divergent differentiation programs. Increasing Nodal signals above basal levels by direct stimulation with Activin promotes differentiation towards the mesendodermal lineages while repression of signaling with the specific Nodal/Activin receptor inhibitor SB431542 induces trophectodermal differentiation. To address how quantitative Nodal/Activin signals are translated qualitatively into distinct cell fates decisions, we performed chromatin immunoprecipitation of phospho-Smad2, the primary downstream transcriptional factor of the Nodal/Activin pathway, followed by massively parallel sequencing, and show that phospho-Smad2 binds to and regulates distinct subsets of target genes in a dose-dependent manner. Crucially, Nodal/Activin signaling directly controls the Oct4 master regulator of pluripotency by graded phospho-Smad2 binding in the promoter region. Hence stem cells interpret and carry out differential Nodal/Activin signaling instructions via a corresponding gradient of Smad2 phosphorylation that selectively titrates self-renewal against alternative differentiation programs by direct regulation of distinct target gene subsets and Oct4 expression.
Nodal and Activin are extracellular signaling molecules that diffuse from the source of secretion and induce recipient stem cells to become new cell types according to a concentration gradient. In the early embryo, they are important for the specification of tissues at the correct place and time, but paradoxically they drive the opposite function in embryonic and epiblast stem cells where they maintain the stem cell state instead of promoting differentiation. The molecular basis of how the level of signaling determines stem cell fate decisions remains poorly understood. We found that Smad2, the main transcription factor of the Nodal/Activin pathway was phosphorylated according to the level of signaling. By mapping where phospho-Smad2 binds in the embryonic stem cell genome and how this affects transcription of the associated target genes, we show that phospho-Smad2 can recruit and regulate different sets of target gene depending on the signaling level. Moreover, phospho-Smad2 also directly regulates Oct4, a master gene controlling the stem cell state thereby reconciling the opposing functions of the Nodal/Activin pathway in differentiation versus self-renewal programs. The pathway can mediate the exit from self-renewal via Oct4 and simultaneously drives differentiation towards particular lineages by recruiting the relevant gene subsets for this purpose.
The mechanisms by which human embryonic stem cells (hESC) differentiate to endodermal lineage have not been extensively studied. Mathematical models can aid in the identification of mechanistic information. In this work we use a population-based modeling approach to understand the mechanism of endoderm induction in hESC, performed experimentally with exposure to Activin A and Activin A supplemented with growth factors (basic fibroblast growth factor (FGF2) and bone morphogenetic protein 4 (BMP4)). The differentiating cell population is analyzed daily for cellular growth, cell death, and expression of the endoderm proteins Sox17 and CXCR4. The stochastic model starts with a population of undifferentiated cells, wherefrom it evolves in time by assigning each cell a propensity to proliferate, die and differentiate using certain user defined rules. Twelve alternate mechanisms which might describe the observed dynamics were simulated, and an ensemble parameter estimation was performed on each mechanism. A comparison of the quality of agreement of experimental data with simulations for several competing mechanisms led to the identification of one which adequately describes the observed dynamics under both induction conditions. The results indicate that hESC commitment to endoderm occurs through an intermediate mesendoderm germ layer which further differentiates into mesoderm and endoderm, and that during induction proliferation of the endoderm germ layer is promoted. Furthermore, our model suggests that CXCR4 is expressed in mesendoderm and endoderm, but is not expressed in mesoderm. Comparison between the two induction conditions indicates that supplementing FGF2 and BMP4 to Activin A enhances the kinetics of differentiation than Activin A alone. This mechanistic information can aid in the derivation of functional, mature cells from their progenitors. While applied to initial endoderm commitment of hESC, the model is general enough to be applicable either to a system of adult stem cells or later stages of ESC differentiation.
Manipulatable models of bladder development which interrogate specific pathways are badly needed. Such models will allow a systematic investigation of the multitude of pathologies which result from developmental defects of the urinary bladder. In the present communication, we describe a model in which mouse embryonic stem (ES) cells are directed to differentiate to form bladder tissue by specific interactions with fetal bladder mesenchyme. This model allows us to visualize the various stages in the differentiation of urothelium from ES cells, including the commitment to an endodermal cell lineage, with the temporal profile characterized by examining the induction of specific endodermal transcription factors (Foxa1 and Foxa2). In addition, final functional urothelial differentiation was characterized by examining uroplakin expression. It is well established that ES cells will spontaneously develop teratomas when grown within immunocompromised mouse hosts. We determined the specific mesenchymal to ES cell ratios necessary to dictate organ-specific differentiation while completely suppressing teratomatous growth. Embryonic mesenchyme is well established as an inductive tissue which dictates organ-specific programming of epithelial tissues. The present study demonstrates that embryonic bladder mesenchyme can also steer ES cells towards developing specific endodermal derived urothelium. These approaches allow us to capture specific stages of stem cell differentiation and to better define stem cell hierarchies.
In the present study, mouse embryonic stem cells (ESCs) were differentiated into alveolar epithelial type II (AEII) cells for endotracheal injection. These enriched lung-like populations expressed lung epithelial markers SP-A, SP-B, SP-C, and CC10. First we show that rapid differentiation of ESCs requires a dissociated seeding method instead of an embryoid body culture method. We then investigated a two-step differentiation of ESCs into definitive endoderm by activin or A549-conditioned medium as a precursor to lung epithelial cells. When conditioned medium from A549 cells was used to derive endoderm, yield was increased above that of activin alone. Further studies showed that Wnt3a may be one of the secreted factors produced by A549 cells and promotes definitive endoderm differentiation, in part, through suppression of primitive endoderm. Activin and Wnt3a together at appropriate doses with dissociated cell seeding promoted greater endoderm yield than activin alone. Next, fibroblast growth factor 2 was shown to induce a dose-dependent expression of SPC, and these cells contained lamellar bodies characteristic of mature AEII cells from ESC-derived endoderm. Finally, ES-derived lung cells were endotracheally injected into preterm mice with evidence of AEII distribution within the lung parenchyma. This study concludes that a recapitulation of development may enhance derivation of an enriched population of lung-like cells for use in cell-based therapy.
Embryoid bodies formed from teratocarcinoma stem cells differentiate an outer layer consisting of parietal and visceral endoderm or of visceral endoderm exclusively. We have previously shown that when these embryoid bodies are plated on collagen-coated substrates a parietal endoderm- like cell migrates onto the substrate, whereas all of the visceral endoderm remains associated with the stem cell mass, suggesting a role for substrate contact in parietal endoderm differentiation. We now identify fibronectin as the migration-promoting component in these cultures, and note that laminin and collagen type IV are 10-fold less effective at promoting both attachment and endoderm outgrowth. The RGDS tetrapeptide (arg-gly-asp-ser) from the cell attachment domain of fibronectin can specifically block attachment and outgrowth on both fibronectin- and laminin-coated substrates. In addition, the involvement of the 140-kD fibronectin receptor is demonstrated using an antibody directed against this molecule.
Single-layered epithelia are the first differentiated cell types to develop in the embryo, with columnar and squamous types appearing immediately after blastocyst implantation. Here, we show that mouse embryonic stem cells seeded on hensin or laminin, but not fibronectin or collagen type IV, formed hemispheric epithelial structures whose outermost layer terminally differentiated to an epithelium that resembled the visceral endoderm. Hensin induced columnar epithelia, whereas laminin formed squamous epithelia. At the egg cylinder stage, the distal visceral endoderm is columnar, and these cells begin to migrate anteriorly to create the anterior visceral endoderm, which assumes a squamous shape. Hensin expression coincided with the dynamic appearance and disappearance of columnar cells at the egg cylinder stage of the embryo. These expression patterns, and the fact that hensin null embryos (and those already reported for laminin) die at the onset of egg cylinder formation, support the view that hensin and laminin are required for terminal differentiation of columnar and squamous epithelial phenotypes during early embryogenesis.
epithelium; extracellular matrix proteins; cell size; embryonic development; stem cell
Pluripotent embryonic stem cells hold a great promise as an unlimited source of tissue for treatment of chronic diseases such as Type 1 diabetes. Herein, we describe a protocol using all-trans-retinoic acid, basic fibroblast growth factor and dibutyryl cAMP (DBcAMP) in the absence of embryoid body formation, for differentiation of murine embryonic stem cells into definitive endoderm that may serve as pancreatic precursors. The produced cells were analyzed by quantitative PCR, immunohistochemistry and static insulin release assay for markers of trilaminar embryo, and pancreas. Differentiated cells displayed increased Sox17 and Foxa2 expression consistent with definitive endoderm production. There was minimal production of Sox7, an extraembryonic endoderm marker, and Oct4, a marker of pluripotency. There was minimal mesoderm or neuroectoderm formation based on expression levels of the markers brachyury and Sox1, respectively. Various assays revealed that the cell clusters generated by this protocol express markers of the pancreatic lineage including insulin I, insulin II, C-peptide, PDX-1, carboxypeptidase E, pan-cytokeratin, amylase, glucagon, PAX6, Ngn3 and Nkx6.1. This protocol using all-trans-retinoic acid, DBcAMP, in the absence of embryoid bodies, generated cells that have features of definitive endoderm that may serve as pancreatic endocrine precursors.
To identify early populations of committed progenitors derived from human embryonic stem cells (hESCs), we screened self-renewing, BMP4-treated and retinoic acid–treated cultures with >400 antibodies recognizing cell-surface antigens. Sorting of >30 subpopulations followed by transcriptional analysis of developmental genes identified four distinct candidate progenitor groups. Subsets detected in self-renewing cultures, including CXCR4+ cells, expressed primitive endoderm genes. Expression of Cxcr4 in primitive endoderm was confirmed in visceral endoderm of mouse embryos. BMP4-induced progenitors exhibited gene signatures of mesoderm, trophoblast and vascular endothelium, suggesting correspondence to gastrulation-stage primitive streak, chorion and allantois precursors, respectively. Functional studies in vitro and in vivo confirmed that ROR2+ cells produce mesoderm progeny, APA+ cells generate syncytiotrophoblasts and CD87+ cells give rise to vasculature. The same progenitor classes emerged during the differentiation of human induced pluripotent stem cells (hiPSCs). These markers and progenitors provide tools for purifying human tissue-regenerating progenitors and for studying the commitment of pluripotent stem cells to lineage progenitors.
Background. Mouse embryonic stem (ES) cells can be differentiated in vitro by aggregation and/or retinoic acid (RA) treatment. The principal differentiation lineage in vitro is extraembryonic primitive endoderm. Dab2, Laminin, GATA4, GATA5, and GATA6 are expressed in embryonic primitive endoderm and play critical roles in its lineage commitment. Results. We found that in the absence of GATA4 or GATA5, RA-induced primitive endoderm differentiation of ES cells was reduced. GATA4 (−/−) ES cells express higher level of GATA5, GATA6, and hepatocyte nuclear factor 4 alpha marker of visceral endoderm lineage. GATA5 (−/−) ES cells express higher level of alpha fetoprotein marker of early liver development. GATA6 (−/−) ES cells express higher level of GATA5 as well as mesoderm and cardiomyocyte markers which are collagen III alpha-1 and tropomyosin1 alpha. Thus, deletion of GATA6 precluded endoderm differentiation but promoted mesoderm lineages. Conclusions. GATA4, GATA5, and GATA6 each convey a unique gene expression pattern and influences ES cell differentiation. We showed that ES cells can be directed to avoid differentiating into primitive endoderm and to adopt unique lineages in vitro by modulating GATA factors. The finding offers a potential approach to produce desirable cell types from ES cells, useful for regenerative cell therapy.
Prior to gastrulation in the mouse, all endodermal cells arise from the primitive
endoderm of the blastocyst stage embryo. Primitive endoderm and its derivatives
are generally referred to as extra-embryonic endoderm (ExEn) because the
majority of these cells contribute to extra-embryonic lineages encompassing the
visceral endoderm (VE) and the parietal endoderm (PE). During gastrulation, the
definitive endoderm (DE) forms by ingression of cells from the epiblast. The DE
comprises most of the cells of the gut and its accessory organs. Despite their
different origins and fates, there is a surprising amount of overlap in marker
expression between the ExEn and DE, making it difficult to distinguish between
these cell types by marker analysis. This is significant for two main reasons.
First, because endodermal organs, such as the liver and pancreas, play important
physiological roles in adult animals, much experimental effort has been directed
in recent years toward the establishment of protocols for the efficient
derivation of endodermal cell types in vitro. Conversely,
factors secreted by the VE play pivotal roles that cannot be attributed to the
DE in early axis formation, heart formation and the patterning of the anterior
nervous system. Thus, efforts in both of these areas have been hampered by a
lack of markers that clearly distinguish between ExEn and DE. To further
understand the ExEn we have undertaken a comparative analysis of three ExEn-like
cell lines (END2, PYS2 and XEN). PYS2 cells are derived from embryonal
carcinomas (EC) of 129 strain mice and have been characterized as parietal
endoderm-like , END2 cells are derived from P19 ECs and
described as visceral endoderm-like, while XEN cells are derived from blastocyst
stage embryos and are described as primitive endoderm-like. Our analysis
suggests that none of these cell lines represent a bona fide
single in vivo lineage. Both PYS2 and XEN cells represent mixed
populations expressing markers for several ExEn lineages. Conversely END2 cells,
which were previously characterized as VE-like, fail to express many markers
that are widely expressed in the VE, but instead express markers for only a
subset of the VE, the anterior visceral endoderm. In addition END2 cells also
express markers for the PE. We extended these observations with microarray
analysis which was used to probe and refine previously published data sets of
genes proposed to distinguish between DE and VE. Finally, genome-wide pathway
analysis revealed that SMAD-independent TGFbeta signaling through a TAK1/p38/JNK
or TAK1/NLK pathway may represent one mode of intracellular signaling shared by
all three of these lines, and suggests that factors downstream of these pathways
may mediate some functions of the ExEn. These studies represent the first step
in the development of XEN cells as a powerful molecular genetic tool to study
the endodermal signals that mediate the important developmental functions of the
extra-embryonic endoderm. Our data refine our current knowledge of markers that
distinguish various subtypes of endoderm. In addition, pathway analysis suggests
that the ExEn may mediate some of its functions through a non-classical MAP
Kinase signaling pathway downstream of TAK1.
The mechanisms controlling thyrocyte development during embryonic stem (ES) cell differentiation have only been partially elucidated, although previous studies have suggested the participation of thyroid stimulating hormone (TSH) in these processes. To further define the role of TSH in this context, we have studied a murine ES cell line in which green fluorescent protein (GFP) cDNA is targeted to the TSH receptor (TSHR) gene, linking the expression of GFP to the transcription of the endogenous TSHR gene. We demonstrate that, in the initial stages of embryoid body formation, activin A and TSH induce the differentiation of definitive endoderm and thyrocyte progenitors expressing Sox17, Foxa2 and TSHR. These thyrocyte progenitors are then converted into cellular aggregates that, in the presence of insulin and IGF-1, further differentiate into mature thyroglobulin-expressing thyrocytes. Our data suggest that, despite the fact that TSH is important for the induction and specification of thyrocytes from ES cells, insulin and IGF-1 are crucial for thyrocyte maturation. Our method provides a powerful in vitro differentiation model for studying the mechanisms of early thyrocyte lineage development.
Embryonic stem cell; thyrocyte; thyroid stimulating hormone; insulin; insulin-like growth factor 1
The endoderm is a multipotent progenitor cell population in the embryo that gives rise to the liver, pancreas, and other cell types and provides paradigms for understanding cell type specification. Studies of isolated embryo tissue cells and genetic approaches in vivo have defined FGF/MAPK and BMP signaling pathways that induce liver and pancreatic fates in the endoderm. In undifferentiated endoderm cells, the FoxA and GATA transcription factors are among the first to engage silent genes, helping to endow competence for cell type specification. FoxA proteins can bind their target sites in highly compacted chromatin and open up the local region for other factors to bind; hence they have been termed "pioneer factors". We recently found that FoxA proteins remain bound to chromatin in mitosis, as an epigenetic mark. In embryonic stem cells, which lack FoxA, FoxA target sites can be occupied by FoxD3, which in turn helps maintain a local demethylation of chromatin. By these means, a cascade of Fox factors helps endow progenitor cells with the competence to activate genes in response to tissue-inductive signals. Understanding such epigenetic mechanisms for transcriptional competence coupled with knowledge of the relevant signals for cell type specification should greatly facilitate efforts to predictably differentiate stem cells to liver and pancreatic fates.
Investigating development of inaccessible human tissues like embryonic endoderm with embryonic stem cell (ESC) has been hindered by a lack of methods for marking and isolating endodermal cells, and tracing fates of their progeny toward differentiated lineages. Using homologous recombination in human ESC, we inserted an enhanced green fluorescent protein (eGFP) transgene into a locus encoding a postulated marker of human endoderm, SOX17, permitting purification of SOX17+ hESC progeny by fluorescence activated cell sorting (FACS). Microarray studies revealed a unique gene expression profile of human SOX17+ cells including endodermal marker enrichment, and unveiled specific cell surface protein combinations that permitted FACS-based isolation of primitive gut tube endodermal cells produced from unmodified human ESCs and from induced pluripotent stem cells (iPSC). FACS-isolated SOX17+ endodermal cells differentiated to progeny expressing markers of liver, pancreas, and intestinal epithelium, providing unprecedented evidence that human gastrointestinal lineages derive from SOX17+ cells. Thus, prospective isolation, lineage tracing, and developmental studies of hESCs described here have revealed fundamental aspects of human endodermal biology.
We previously reported that chick anterolateral endoderm (AL endoderm) induces cardiomyogenesis in mouse embryoid bodies. However, the requirement to micro-dissect AL endoderm from gastrulation-stage embryos precludes its use to identify novel cardiomyogenic factors, or to scale up cardiomyocyte numbers for therapeutic experiments. To circumvent this problem we have addressed whether human definitive endoderm (hDE) cells, which can be efficiently generated in large numbers from human embryonic stem cells (hESCs), can mimic the ability of AL endoderm to induce cardiac myogenesis. Results demonstrate that both hDE cells and medium conditioned by them induce cardiac myogenesis in pluripotent hESCs, as indicated by rhythmic beating and immunohistochemical/quantitative polymerase chain reaction monitoring of marker gene expression. The cardiomyogenic effect of hDE is enhanced when pluripotent hESCs are preinduced to the mes-endoderm state. Because this approach is tractable and scalable, it may facilitate identification of novel hDE-secreted factors for inclusion in defined cardiomyogenic cocktails.
Wnt signaling has been implicated in many developmental processes, but its role in early endoderm development is not well understood. Wnt signaling is active in posterior endoderm as early as E7.5. Genetic and chemical activation show that the Wnt pathway acts directly on endoderm to induce the intestinal master regulator Cdx2, shifting global gene away from anterior endoderm and toward a posterior, intestinal program. In a mouse embryonic stem cell differentiation platform that yields pure populations of definitive endoderm, Wnt signaling induces intestinal gene expression in all cells. We have identified a set of genes specific to the anterior small intestine, posterior small intestine, and large intestine during early development, and show that Wnt, through Cdx2, activates large intestinal gene expression at high doses and small intestinal gene expression at lower doses. These findings shed light on the mechanism of embryonic intestinal induction and provide a method to manipulate intestinal development from embryonic stem cells.
Endoderm; Embryonic stem cell differentiation; Intestinal development
Genetic inactivation of the transcription factor GATA-6 in the embryoid body induces massive apoptosis at the early stage of ES cell differentiation. Evidence is provided that BMP-2 is a direct transcription target of GATA-6 and mediates GATA-6-dependent cell survival in concert with endoderm-derived basement membrane.
GATA-6 is a zinc-finger transcription factor essential for early embryogenesis. Ablation of GATA-6 in mice impairs endoderm differentiation and causes apoptosis of epiblast cells. The endoderm defects have been attributed to the loss of HNF4, disabled-2, and GATA-4. However, the mechanisms underlying epiblast apoptosis are unclear. In this study we used mouse embryonic stem cell–derived embryoid bodies (EBs) as a model for peri-implantation development and found that ablation of GATA-6 causes massive apoptosis during EB differentiation. Endoderm grafting experiments and ectopic basement membrane (BM) assembly suggest that both BM and non-BM factors contribute to cell survival. Furthermore, the increased cell death in mutant EBs is accompanied by reduced expression of bone morphogenetic protein 2 (BMP-2). Chromatin immunoprecipitation reveals direct binding of GATA-6 to the Bmp2 promoter. Treatment of the mutant EBs with BMP-2 markedly suppresses apoptosis, whereas stable overexpression of the BMP antagonist noggin or a dominant-negative BMP receptor in normal EBs leads to increased apoptosis. Last, activation of SMAD1/5 by phosphorylation is significantly inhibited in the absence of GATA-6, and this is reversed by exogenous BMP-2. Treatment of normal EBs with SMAD phosphorylation inhibitor increases apoptosis. Collectively these results suggest that GATA-6 promotes cell survival by regulating endoderm expression of BMP-2 and BM during embryonic epithelial morphogenesis.
We have previously shown that the inhibition of fibroblast growth factor (FGF) signaling induced endodermal gene expression in the animal cap and caused the expansion of the endodermal mass in Xenopus embryos. However, we still do not know whether or not the alteration of FGF signaling controls embryonic cell fate, or when FGF signal blocking is required for endoderm formation in Xenopus. Here, we show that FGF signal blocking in embryonic cells causes their descendants to move into the endodermal region and to express endodermal genes. It is also interesting that blocking FGF signaling between fertilization and embryonic stage 10.5 promotes endoderm formation, but persistent FGF signaling blocking after stage 10.5 restricts endoderm formation and differentiation.
embryonic development; endoderm; fibroblast growth factors; SU 5402; Xenopus
Embryonic stem cell (ESC) derivatives offer promise for generating clinically useful tissues for transplantation, yet the specter of producing tumors in patients remains a significant concern. We have developed a simple method that eliminates the tumorigenic potential from differentiated ESC cultures of murine and human origin while purifying lineage-restricted, definitive endoderm-committed cells. A three-stage scheme utilizing magnetic bead sorting and specific antibodies to remove undifferentiated ESCs and extraembryonic endoderm cells, followed by positive selection of definitive endoderm cells on the basis of epithelial cell adhesion molecule (EpCAM) expression, was used to isolate a population of EpCAM+SSEA1−SSEA3− cells. Sorted cells do not form teratomas after transplantation into immunodeficient mice, but display gene and protein expression profiles indicative of definitive endoderm cells. Sorted cells could be subsequently expanded in vitro and further differentiated to express key pancreas specification proteins. In vivo transplantation of sorted cells resulted in small, benign tissues that uniformly express PDX1. These studies describe a straightforward method without genetic manipulation that eliminates the risk of teratoma formation from ESC differentiated derivatives. Significantly, enriched populations isolated by this method appear to be lineage-restricted definitive endoderm cells with limited proliferation capacity.
Embryonic stem (ES) cells are a potential source of a variety of differentiated cells for cell therapy, drug discovery, and toxicology screening. Here, we present an efficacy strategy for the differentiation of mouse ES cells into insulin-producing cells (IPCs) by a two-step differentiation protocol comprising of (i) the formation of definitive endoderm in monolayer culture by activin A, and (ii) this monolayer endoderm being induced to differentiate into IPCs by nicotinamide, insulin, and laminin. Differentiated cells can be obtained within approximately 7 days. The differentiation IPCs combined application of RT-PCR, ELISA, and immunofluorescence to characterize phenotypic and functional properties. In our study, we demonstrated that IPCs produced pancreatic transcription factors, endocrine progenitor marker, definitive endoderm, pancreatic β-cell markers, and Langerhans α and δ cells. The IPCs released insulin in a manner that was dose dependent upon the amount of glucose added. These techniques may be able to be applied to human ES cells, which would have very important ramifications for treating human disease.
Interactions between the endoderm and mesoderm that mediate myocardial induction are difficult to study in vivo because of the small size of mammalian embryos at relevant stages. However, we and others have demonstrated that signals from endodermal cell lines can influence myocardial differentiation from both mouse and human embryoid bodies (EBs), and because of this, assays that utilize embryonic stem (ES) cells and endodermal cell lines provide excellent in vitro models to study early cardiac differentiation. Extraembryonic endoderm (XEN) stem cells have a particular advantage over other heart-inducing cell lines in that they can easily be derived from both wild type and mutant mouse blastocysts. Here we describe the first isolation of a Nodal mutant XEN stem cell line. Nodal−/− XEN cell lines were not isolated at expected Mendelian ratios, and those that were successfully established, showed an increase in markers for the anterior visceral endoderm (AVE). Since AVE represents the heart-inducing endoderm in the mouse, cardiac differentiation was compared in EBs treated with conditioned medium (CM) collected from wild type or Nodal−/− XEN cells. EBs treated with CM from Nodal−/− cells began beating earlier and showed early activation of myocardial genes, but this early cardiac differentiation did not cause an overall increase in cardiomyocyte yield. By comparison, CM from wild type XEN cells both delayed cardiac differentiation and caused a concomitant increase in overall cardiomyocyte formation. Detailed marker analysis suggested that early activation of cardiac differentiation by Nodal−/− XEN CM caused premature differentiation and subsequent depletion of cardiac progenitors.
Nodal; Extraembryonic endoderm (XEN) stem cells; Cardiomyocyte differentiation; Mouse ES cells