The study of how human embryonic stem cells (hESCs) differentiate into insulin-producing beta cells has two-fold significance: first, it provides an in vitro model system for the study of human pancreatic development, and second, it serves as a platform for the ultimate production of beta cells for transplantation into patients with diabetes. The delineation of growth factor interactions regulating pancreas specification from hESCs in vitro is critical to achieving these goals. In this study, we describe the roles of growth factors bFGF, BMP4 and Activin A in early hESC fate determination. The entire differentiation process is carried out in serum-free chemically defined media (CDM) and results in reliable and robust induction of pancreatic endoderm cells, marked by PDX1, and cell clusters co-expressing markers characteristic of beta cells, including PDX1 and insulin/C-peptide. Varying the combinations of growth factors, we found that treatment of hESCs with bFGF, Activin A and BMP4 (FAB) together for 3 to 4 days resulted in strong induction of primitive-streak and definitive endoderm-associated genes, including MIXL1, GSC, SOX17 and FOXA2. Early proliferative foregut endoderm and pancreatic lineage cells marked by PDX1, FOXA2 and SOX9 expression are specified in EBs made from FAB-treated hESCs, but not from Activin A alone treated cells. Our results suggest that important tissue interactions occur in EB-based suspension culture that contribute to the complete induction of definitive endoderm and pancreas progenitors. Further differentiation occurs after EBs are embedded in Matrigel and cultured in serum-free media containing insulin, transferrin, selenium, FGF7, nicotinamide, islet neogenesis associated peptide (INGAP) and exendin-4, a long acting GLP-1 agonist. 21–28 days after embedding, PDX1 gene expression levels are comparable to those of human islets used for transplantation, and many PDX1+ clusters are formed. Almost all cells in PDX1+ clusters co-express FOXA2, HNF1ß, HNF6 and SOX9 proteins, and many cells also express CPA1, NKX6.1 and PTF1a. If cells are then switched to medium containing B27 and nicotinamide for 7 to 14 days, then the number of insulin+ cells increases markedly. Our study identifies a new chemically defined culture protocol for inducing endoderm- and pancreas-committed cells from hESCs and reveals an interplay between FGF, Activin A and BMP signaling in early hESC fate determination.
definitive endoderm; beta cell; bone morphogenetic protein; fibroblast growth factor; human embryonic stem cells; Activin A
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.
Lineage specific differentiation of human embryonic stem cells (hESCs) is largely mediated by specific growth factors and extracellular matrix molecules. Growth factors initiate a cascade of signals which control gene transcription and cell fate specification. There is a lot of interest in inducing hESCs to an endoderm fate which serves as a pathway towards more functional cell types like the pancreatic cells. Research over the past decade has established several robust pathways for deriving endoderm from hESCs, with the capability of further maturation. However, in our experience, the functional maturity of these endoderm derivatives, specifically to pancreatic lineage, largely depends on specific pathway of endoderm induction. Hence it will be of interest to understand the underlying mechanism mediating such induction and how it is translated to further maturation. In this work we analyze the regulatory interactions mediating different pathways of endoderm induction by identifying co-regulated transcription factors.
hESCs were induced towards endoderm using activin A and 4 different growth factors (FGF2 (F), BMP4 (B), PI3KI (P), and WNT3A (W)) and their combinations thereof, resulting in 15 total experimental conditions. At the end of differentiation each condition was analyzed by qRT-PCR for 12 relevant endoderm related transcription factors (TFs). As a first approach, we used hierarchical clustering to identify which growth factor combinations favor up-regulation of different genes. In the next step we identified sets of co-regulated transcription factors using a biclustering algorithm. The high variability of experimental data was addressed by integrating the biclustering formulation with bootstrap re-sampling to identify robust networks of co-regulated transcription factors. Our results show that the transition from early to late endoderm is favored by FGF2 as well as WNT3A treatments under high activin. However, induction of late endoderm markers is relatively favored by WNT3A under high activin.
Use of FGF2, WNT3A or PI3K inhibition with high activin A may serve well in definitive endoderm induction followed by WNT3A specific signaling to direct the definitive endoderm into late endodermal lineages. Other combinations, though still feasible for endoderm induction, appear less promising for pancreatic endoderm specification in our experiments.
Human embryonic stem cells; Endoderm; Hierarchical clustering; Biclustering; Bootstrap
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.
Detection of low-level, lineage-specific transcription aids in the identification of lineage-primed populations of ES cells provides a new framework for pluripotency.
ES cells are defined as self-renewing, pluripotent cell lines derived from early embryos. Cultures of ES cells are also characterized by the expression of certain markers thought to represent the pluripotent state. However, despite the widespread expression of key markers such as Oct4 and the appearance of a characteristic undifferentiated morphology, functional ES cells may represent only a small fraction of the cultures grown under self-renewing conditions. Thus phenotypically “undifferentiated” cells may consist of a heterogeneous population of functionally distinct cell types. Here we use a transgenic allele designed to detect low level transcription in the primitive endoderm lineage as a tool to identify an immediate early endoderm-like ES cell state. This reporter employs a tandem array of internal ribosomal entry sites to drive translation of an enhanced Yellow Fluorescent Protein (Venus) from the transcript that normally encodes for the early endodermal marker Hex. Expression of this Venus transgene reports on single cells with low Hex transcript levels and reveals the existence of distinct populations of Oct4 positive undifferentiated ES cells. One of these cells types, characterized by both the expression of the Venus transgene and the ES cells marker SSEA-1 (V+S+), appears to represent an early step in primitive endoderm specification. We show that the fraction of cells present within this state is influenced by factors that both promote and suppress primitive endoderm differentiation, but conditions that support ES cell self-renewal prevent their progression into differentiation and support an equilibrium between this state and at least one other that resembles the Nanog positive inner cell mass of the mammalian blastocysts. Interestingly, while these subpopulations are equivalently and clonally interconvertible under self-renewing conditions, when induced to differentiate both in vivo and in vitro they exhibit different behaviours. Most strikingly when introduced back into morulae or blastocysts, the V+S+ population is not effective at contributing to the epiblast and can contribute to the extra-embryonic visceral and parietal endoderm, while the V−S+ population generates high contribution chimeras. Taken together our data support a model in which ES cell culture has trapped a set of interconvertible cell states reminiscent of the early stages in blastocyst differentiation that may exist only transiently in the early embryo.
Embryonic stem (ES) cells are karyotypically normal, embryo-derived cell lines that are pluripotent, i.e. capable of generating all the cell types of the future organism, but not the extra-embryonic lineages. What gives ES cells this unique capacity? Here, we use a fluorescent reporter cell line that employs translational amplification to visualize single ES cells expressing low levels of lineage-specific genes. With this reporter we split ES cell cultures into two fractions that both express certain stem cell markers but only one of which expresses low levels of an endodermal marker gene. Following purification, single cells from either fraction are equally competent to re-establish a heterogeneous culture. However, when challenged to differentiate immediately after purification, each exhibits strong lineage bias, with the endoderm marker-expressing fraction unexpectedly able to contribute to the extra-embryonic endoderm in chimeric embryos. These data suggest that ES cells expand under steady-state conditions as a heterogeneous mix of lineage-biased—but not lineage-committed—cell types. We propose that these observed uncommitted substates exist temporarily in vivo, but are perpetuated in vitro under the selectively self-renewing conditions of ES cell culture. Our findings suggest that pluripotency is determined by the capacity of a mixed population of lineage-biased intermediates to commit to different cell fates in specific contexts.
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
Liver transplantation is the only definitive treatment for end-stage cirrhosis and fulminant liver failure, but the lack of available donor livers is a major obstacle to liver transplantation. Recently, induced pluripotent stem cells (iPSCs) derived from the reprogramming of somatic fibroblasts, have been shown to resemble embryonic stem (ES) cells in that they have pluripotent properties and the potential to differentiate into all cell lineages in vitro, including hepatocytes. Thus, iPSCs could serve as a favorable cell source for a wide range of applications, including drug toxicity testing, cell transplantation, and patient-specific disease modeling. Here, we describe an efficient and rapid three-step protocol that is able to rapidly generate hepatocyte-like cells from human iPSCs. This occurs because the endodermal induction step allows for more efficient and definitive endoderm cell formation. We show that hepatocyte growth factor (HGF), which synergizes with activin A and Wnt3a, elevates the expression of the endodermal marker Foxa2 (forkhead box a2) by 39.3% compared to when HGF is absent (14.2%) during the endodermal induction step. In addition, iPSC-derived hepatocytes had a similar gene expression profile to mature hepatocytes. Importantly, the hepatocyte-like cells exhibited cytochrome P450 3A4 (CYP3A4) enzyme activity, secreted urea, uptake of low-density lipoprotein (LDL), and possessed the ability to store glycogen. Moreover, the hepatocyte-like cells rescued lethal fulminant hepatic failure in a nonobese diabetic severe combined immunodeficient mouse model. Conclusion: We have established a rapid and efficient differentiation protocol that is able to generate functional hepatocyte-like cells from human iPSCs. This may offer an alternative option for treatment of liver diseases.
Expression of NKX2-1 is required to specify definitive endoderm to respiratory endoderm. However, the transcriptional regulation of NKX2-1 is not fully understood. Here we demonstrate that aside from specifying undifferentiated human embryonic stem cell (hESC) to definitive endoderm, high concentrations of Activin-A are also necessary and sufficient to induce hESC-derived definitive endodermal progeny to a FOXA2/NKX2-1/GATA6/PAX9 positive respiratory epithelial fate. Activin-A directly mediates the induction of NKX2-1 by interacting with ALK4, leading to phosphorylation of SMAD2, which binds directly to the NKX2-1 promoter and activates its expression. Activin-A can be replaced by GDF11 but not transforming growth factor β1. Addition of Wnt3a, SHH, FGF2, or BMP4 failed to induce NKX2-1. These results suggest that direct binding of Activin-A–responsive SMAD2 to the NKX2-1 promoter plays essential role during respiratory endoderm specification.
In vitro production of a definitive endoderm (DE) is an important issue in stem
cell-related differentiation studies and it can assist with the production of more efficient
endoderm derivatives for therapeutic applications. Despite tremendous progress in DE
differentiation of human embryonic stem cells (hESCs), researchers have yet to discover
universal, efficient and cost-effective protocols.
Materials and Methods:
In this experimental study, we have treated hESCs with 200 nM of
Stauprimide (Spd) for one day followed by activin A (50 ng/ml; A50) for the next three days (Spd-
A50). In the positive control group, hESCs were treated with Wnt3a (25 ng/ml) and activin A (100
ng/ml) for the first day followed by activin A for the next three days (100 ng/ml; W/A100-A100).
Gene expression analysis showed up regulation of DE-specific marker genes
(SOX17, FOXA2 and CXCR4) comparable to that observed in the positive control group. Expression
of the other lineage specific markers did not significantly change (p<0.05). We also
obtained the same gene expression results using another hESC line. The use of higher concentrations
of Spd (400 and 800 nM) in the Spd-A50 protocol caused an increase in the expression
SOX17 as well as a dramatic increase in mortality rate of the hESCs. A lower concentration
of activin A (25 ng/ml) was not able to up regulate the DE-specific marker genes. Then, A50 was
replaced by inducers of definitive endoderm; IDE1/2 (IDE1 and IDE2), two previously reported
small molecule (SM) inducers of DE, in our protocol (Spd-IDE1/2). This replacement resulted
in the up regulation of visceral endoderm (VE) marker (SOX7) but not DE-specific markers.
Therefore, while the Spd-A50 protocol led to DE production, we have shown that IDE1/2 could
not fully replace activin A in DE induction of hESCs.
These findings can assist with the design of more efficient chemically-defined
protocols for DE induction of hESCs and lead to a better understanding of the different
signaling networks that are involved in DE differentiation of hESCs.
Definitive Endoderm; Embryonic Stem Cells; Differentiation; Activin A; Stauprimide
Retinoic acid (RA) and fibroblast growth factor 4 (FGF4) signaling control endoderm patterning and pancreas induction/expansion. Based on these findings, RA and FGFs, excluding FGF4, have frequently been used in differentiation protocols to direct differentiation of hESCs into endodermal and pancreatic cell types. In vivo, these signaling pathways act in a temporal and concentration-dependent manner. However, in vitro, the underlying basis for the time of addition of growth and differentiation factors (GDFs), including RA and FGFs, as well as the concentration is lacking. Thus, in order to develop robust and reliable differentiation protocols of ESCs into mature pancreatic cell types, including insulin-producing β cells, it will be important to mechanistically understand each specification step. This includes differentiation of mesendoderm/definitive endoderm into foregut endoderm- the origin of pancreatic endoderm.
Here, we provide data on the individual and combinatorial role of RA and FGF4 in directing differentiation of ActivinA (AA)-induced hESCs into PDX1-expressing cells. FGF4's ability to affect endoderm patterning and specification in vitro has so far not been tested. By testing out the optimal concentration and timing of addition of FGF4 and RA, we present a robust differentiation protocol that on average generates 32% PDX1+ cells. Furthermore, we show that RA is required for converting AA-induced hESCs into PDX1+ cells, and that part of the underlying mechanism involves FGF receptor signaling. Finally, further characterization of the PDX1+ cells suggests that they represent foregut endoderm not yet committed to pancreatic, posterior stomach, or duodenal endoderm.
In conclusion, we show that RA and FGF4 jointly direct differentiation of PDX1+ foregut endoderm in a robust and efficient manner. RA signaling mediated by the early induction of RARβ through AA/Wnt3a is required for PDX1 expression. Part of RA's activity is mediated by FGF signaling.
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.
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.
Mouse embryonic stem cells (mESC) have been used to study lineage specification in vitro, including towards a hepatocyte-like fate, and such investigations guided lineage differentiation protocols for human (h)ESC. We recently described a four-step protocol to induce hepatocyte-like cells from hESC which also induced hepatocyte-like cell differentiation of mouse induced pluripotent stem cells. As ESC also spontaneously generate hepatocyte-like cells, we here tested whether the growth factors and serum used in this protocol are required to commit mESC and hESC to hepatocyte-like cells. Culture of mESC from two different mouse strains in the absence of serum and growth factors did not induce primitive streak/definitive endoderm genes but induced default differentiation to neuroectoderm on day 6. Although Activin-A and Wnt3 induced primitive streak/definitive endoderm transcripts most robustly in mESC, simple addition of serum also induced these transcripts. Expression of hepatoblast genes occurred earlier when growth factors were used for mESC differentiation. However, further maturation towards functional hepatocyte-like cells was similar in mESC progeny from cultures with serum, irrespective of the addition of growth factors, and irrespective of the mouse strain. This is in contrast to hESC, where growth factors are required for specification towards functional hepatocyte-like cells. Culture of mESC with serum but without growth factors did not induce preferential differentiation towards primitive endoderm or neuroectoderm. Thus, although induction of primitive streak/definitive endoderm specific genes and proteins is more robust when mESC are exposed to a combination of serum and exogenous growth factors, ultimate generation of hepatocyte-like cells from mESC occurs equally well in the presence or absence of exogenous growth factors. The latter is in contrast to what we observed for hESC. These results suggest that differences exist between lineage specific differentiation potential of mESC and hESC, requiring optimization of different protocols for ESC from either species.
During embryonic development signalling pathways act repeatedly in different contexts to pattern the emerging germ layers. Understanding how these different responses are regulated is a central question for developmental biology. In this study, we used mouse embryonic stem cell (mESC) differentiation to uncover a new mechanism for PI3K signalling that is required for endoderm specification. We found that PI3K signalling promotes the transition from naïve endoderm precursors into committed anterior endoderm. PI3K promoted commitment via an atypical activity that delimited epithelial-to-mesenchymal transition (EMT). Akt1 transduced this activity via modifications to the extracellular matrix (ECM) and appropriate ECM could itself induce anterior endodermal identity in the absence of PI3K signalling. PI3K/Akt1-modified ECM contained low levels of Fibronectin (Fn1) and we found that Fn1 dose was key to specifying anterior endodermal identity in vivo and in vitro. Thus, localized PI3K activity affects ECM composition and ECM in turn patterns the endoderm.
From conception to birth, a single fertilised egg will multiply into trillions of cells, with each cell becoming one of the 200 or so different types of cell that are found in the human body. The development of an embryo is complex and dynamic, with cells giving up their ability to become any cell type and committing to becoming a specific cell type within a given tissue. At the same time, different groups of cells migrate to the appropriate locations within the developing embryo. Although it is challenging to decipher the roles of the individual signalling pathways that control an embryo’s development, several important components have been found.
Fibroblast growth factor (FGF) is a protein that regulates the formation of the endoderm: this is the innermost of the three layers of cells that form in the early embryo, and it gives rise to internal organs such as the gut, liver and pancreas. As well as ‘telling’ cells to become the front part, or anterior, of the endoderm, FGF also controls the migration of these cells within the embryo. However, uncoupling these two roles has been a major challenge, and the molecular mechanisms behind them are unclear.
Now, Villegas et al. have discovered that FGF activates a signalling cascade involving two enzymes called PI3K and Akt1. In lab-grown embryonic stem cells—cells that can be coaxed to become any of the cell types formed during development—this signalling cascade is essential for FGF to trigger differentiation of the cell types found in the anterior endoderm. The PI3K/Akt1 signalling cascade achieves this by reducing the level of a protein called fibronectin in the ‘extracellular matrix’ that surrounds the cells. This low level of fibronectin will in turn induce cells to stick together in an organized layer; and this rearrangement of cell-cell and cell-matrix interactions appears linked to triggering the differentiation of anterior endoderm cell types.
Villegas et al. showed that the PI3K/Akt1 pathway was also essential for endoderm formation in living mouse embryos. As a normal embryo develops, the anterior endoderm cells move into a ‘groove’ at the front the embryo, where the level of fibronectin is lower than it is at the posterior end of the embryo.
These findings highlight the importance of the extracellular matrix in the regulation of embryonic development, and should assist in the effort to turn lab-grown stem cells into the useful cell types found in internal organs.
extracellular matrix; Fibronectin; PI3K/Akt1; endoderm; proteomics; foregut; Mouse
Reciprocal interactions between two extra-embryonic tissues, the extra-embryonic ectoderm and the visceral endoderm, and the pluripotent epiblast, are required for the establishment of anterior-posterior polarity in the mouse. After implantation, two visceral endoderm cell types can be distinguished, in the embryonic and extra-embryonic regions of the egg cylinder. In the embryonic region, the specification of the anterior visceral endoderm (AVE) is central to the process of anterior-posterior patterning. Despite recent advances in our understanding of the molecular interactions underlying the differentiation of the visceral endoderm, little is known about how cells colonise the three regions of the tissue.
As a first step, we performed morphological observations to understand how the extra-embryonic region of the egg cylinder forms from the blastocyst. Our analysis suggests a new model for the formation of this region involving cell rearrangements such as folding of the extra-embryonic ectoderm at the early egg cylinder stage. To trace visceral endoderm cells, we microinjected mRNAs encoding fluorescent proteins into single surface cells of the inner cell mass of the blastocyst and analysed the distribution of labelled cells at E5.0, E5.5 and E6.5. We found that at E5.0 the embryonic and extra-embryonic regions of the visceral endoderm do not correspond to distinct cellular compartments. Clusters of labelled cells may span the junction between the two regions even after the appearance of histological and molecular differences at E5.5. We show that in the embryonic region cell dispersion increases after the migration of the AVE. At this time, visceral endoderm cell clusters tend to become oriented parallel to the junction between the embryonic and extra-embryonic regions. Finally we investigated the origin of the AVE and demonstrated that this anterior signalling centre arises from more than a single precursor between E3.5 and E5.5.
We propose a new model for the formation of the extra-embryonic region of the egg cylinder involving a folding of the extra-embryonic ectoderm. Our analyses of the pattern of labelled visceral endoderm cells indicate that distinct cell behaviour in the embryonic and extra-embryonic regions is most apparent upon AVE migration. We also demonstrate the polyclonal origin of the AVE. Taken together, these studies lead to further insights into the formation of the extra-embryonic tissues as they first develop after implantation.
The aim of this study was to assess the impact of transcriptional induction on thyroid follicular cell (TFC) differentiation from endodermally matured embryonic stem (ES) cells. The thyroid transcription factors—NKx2 homeobox 1 (NKx2-1, formerly called TTF-1) and Paired box gene 8 (Pax8)—are known to associate biochemically and synergistically in the activation of thyroid functional genes including the sodium/iodide symporter (NIS), thyrotropin (TSH) receptor (TSHR), thyroglobulin (Tg), and thyroid peroxidase (TPO) genes. In this study, we investigated the ability of ectopically expressed Pax8 and NKx2-1 to further the induction and differentiation of murine ES cells into potential TFCs.
ES cells were stably transfected with either the Pax8 gene, the NKx2-1 gene, or both genes to study the induction of NIS, TSHR, Tg, and TPO genes as assessed using both quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and protein expression. The derived cells were cultured with or without the presence of activin A to allow their differentiation into multipotent endodermal cells.
The four thyroid-specific genes NIS, TSHR, Tg, and TPO were all significantly activated by expressing both transcription factors within the same ES cell. In contrast, significant but much lower transcriptional activity of the TSHR, Tg, and TPO genes was detected in cells expressing just NKx2-1, and only the NIS and TSHR genes responded to Pax8 alone. No Tg protein expression could be detected prior to their development into endodermal derivatives. However, after further differentiation of postembryoid body ES cells with activin A and TSH into endodermal cell lines, those cells with dual transfection of Pax8 and NKx2-1 demonstrated greatly enhanced expression of the NIS, TSHR, Tg, and TPO genes to such a degree that it was similar to that found in control thyroid cells. Furthermore, these same cells formed three-dimensional neofollicles in vitro and expressed Tg protein, but these phenomena were absent from lines expressing only Pax8 or NKx2-1.
These findings provide further evidence that co-expression of Pax8 and NKx2-1 in murine ES cells may induce the differentiation of thyroid-specific gene expression within endodermally differentiated ES cells and commit them to form three-dimensional neofollicular structures.
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
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.
Regeneration is widespread, but mechanisms that activate regeneration remain mysterious. Planarians are capable of whole-body regeneration and mount distinct molecular responses to wounds that result in tissue absence and those that do not. A major question is how these distinct responses are activated. We describe a follistatin homolog (Smed-follistatin) required for planarian regeneration. Smed-follistatin inhibition blocks responses to tissue absence but does not prevent normal tissue turnover. Two activin homologs (Smed-activin-1 and Smed-activin-2) are required for the Smed-follistatin phenotype. Finally, Smed-follistatin is wound-induced and expressed at higher levels following injuries that cause tissue absence. These data suggest that Smed-follistatin inhibits Smed-Activin proteins to trigger regeneration specifically following injuries involving tissue absence and identify a mechanism critical for regeneration initiation, a process important across the animal kingdom.
Most animals can respond to injury with some form of tissue regeneration. In mammals, this is limited to wound healing, whereas other vertebrates—such as salamanders and zebrafish—can regenerate parts of internal organs and even entire appendages. The planarian, a flatworm, is even more remarkable, being able to regenerate its head or tail following amputation, and even a whole animal from just a small body fragment. This is particularly impressive given that planarians have a complex internal anatomy, which includes muscles, intestines, a system similar to kidneys, and a central nervous system with a brain.
How is such regeneration accomplished? Why are planarians able to regenerate their bodies so extensively, whereas humans cannot? To what extent are the mechanisms of planarian regeneration common to other animals? These questions have driven the study of planarian regeneration for more than a century, but it is only in recent years that the tools needed to address these questions at the molecular level have become available.
Planarian regeneration proceeds over several days and involves multiple processes, including gene expression, cell division and cell death. Importantly, it has recently been shown that planarians activate different responses depending on whether an injury results in significant tissue loss—and therefore requires regeneration for repair—or if simple wound healing will be sufficient. The mechanisms behind these different responses to injury have, however, remained a mystery.
Now, Gaviño et al. have identified a key mechanism in the initiation of regeneration following tissue loss. This is centered on the gene follistatin, which is expressed following wounding. When genetic techniques are used to disrupt the expression of follistatin, regeneration is completely blocked. However, the animal’s ability to routinely replace old cells via a stem-cell mediated mechanism is unaffected. This indicates that follistatin is specifically required for the replacement of cells lost through injury. Gaviño et al. further demonstrate that the protein encoded by follistatin likely initiates tissue regeneration upon substantial tissue loss through inhibition of proteins called Activins.
Activin and Follistatin proteins are broadly conserved in evolution, and are also expressed in mammals, raising the possibility that similar molecular circuits may govern regenerative responses in many species.
planarian regeneration; wound signaling; Follistatin; Activin; Other
Here we examine how BMP, Wnt, and FGF signaling modulate activin-induced mesendodermal differentiation of mouse ES cells grown under defined conditions in adherent monoculture. We monitor ES cells containing reporter genes for markers of primitive streak (PS) and its progeny and extend previous findings on the ability of increasing concentrations of activin to progressively induce more ES cell progeny to anterior PS and endodermal fates. We find that the number of Sox17- and Gsc-expressing cells increases with increasing activin concentration while the highest number of T-expressing cells is found at the lowest activin concentration. The expression of Gsc and other anterior markers induced by activin is prevented by treatment with BMP4, which induces T expression and subsequent mesodermal development. We show that canonical Wnt-signaling is required only during late stages of activin-induced development of Sox17-expressing endodermal cells. Furthermore, Dkk1 treatment is less effective in reducing development of Sox17+ endodermal cells in adherent culture than in aggregate culture and appears to inhibit nodal-mediated induction of Sox17+ cells more effectively than activin-mediated induction. Notably, activin-induction of Gsc-GFP+ cells appears refractory to inhibition of canonical Wnt signaling but shows a dependence on early as well as late FGF signaling. Additionally, we find a late dependence on FGF signaling during induction of Sox17+ cells by activin while BMP4-induced T expression requires FGF signaling in adherent but not aggregate culture. Lastly, we demonstrate that activin-induced definitive endoderm derived from mouse ES cells can incorporate into the developing foregut endoderm in vivo and adopt a mostly anterior foregut character after further culture in vitro.
Embryonic stem cell; gastrulation; endoderm; mesendoderm; anterior-posterior patterning; TGF-β; Wnt; FGF
Endoderm formation in the mammalian embryo occurs first in the blastocyst, when the primitive endoderm and pluripotent cells resolve into separate lineages, and again during gastrulation, when the definitive endoderm progenitor population emerges from the primitive streak. The formation of the definitive endoderm can be modeled using pluripotent cell differentiation in culture. The differentiation of early primitive ectoderm-like (EPL) cells, a pluripotent cell population formed from embryonic stem (ES) cells, was used to identify and characterize definitive endoderm formation. Expression of serine peptidase inhibitor, Kazal type 3 (Spink3) was detected in EPL cell–derived endoderm, and in a band of endoderm immediately distal to the embryonic–extra-embryonic boundary in pregastrula and gastrulating embryos. Later expression marked a region of endoderm separating the yolk sac from the developing gut. In the embryo, Spink3 expression marked a region of endoderm comprising the distal visceral endoderm, as determined by an endocytosis assay, and the proximal region of the definitive endoderm. This region was distinct from the more distal definitive endoderm population, marked by thyrotropin-releasing hormone (Trh). Endoderm expressing either Spink3 or Trh could be formed during EPL cell differentiation, and the prevalence of these populations could be influenced by culture medium and growth factor addition. Moreover, further differentiation suggested that the potential of these populations differed. These approaches have revealed an unexpected complexity in the definitive endoderm lineage, a complexity that will need to be accommodated in differentiation protocols to ensure the formation of the appropriate definitive endoderm progenitor in the future.
cellular biology; developmental biology; stem cells
The transcription factor Gata4 is essential for normal heart morphogenesis and regulates the survival, growth, and proliferation of cardiomyocytes. We tested if Gata4 can specify cardiomyocyte fate from an uncommitted stem or progenitor cell population, by developing a system for conditional expression of Gata4 in embryonic stem cells. We find that in embryoid body cultures containing even a low ratio of these cells, expression of Gata4 is sufficient to enhance significantly the generation of cardiomyocytes, via a non-cell-autonomous mechanism. The Gata4-expressing cells do not generate cardiac or other mesoderm derivatives. Rather, Gata4 expression directs the development of two types of Sox17+ endoderm. This includes an epCam+Dpp4+ subtype of visceral endoderm. In addition, Gata4 generates similar amounts of epCam+Dpp4− definitive endoderm enriched for Cxcr4, FoxA2, FoxA3, Dlx5 and other characteristic transcripts. Both types of endoderm express cardiac-inducing factors, including WNT antagonists Dkk1 and Sfrp5, although the visceral endoderm subtype has much higher cardiac inducing activity correlating with relatively enhanced levels of transcripts encoding BMPs. The Gata4-expressing cells eventually express differentiation markers showing commitment to liver development, even under conditions that normally support mesoderm development. The results suggest that Gata4 is capable of specifying endoderm fates that facilitate, with temporal and spatial specificity, the generation of cardiomyocyte progenitors from associated mesoderm.
cardiogenesis; specification; progenitors; WNT; DKK1; BMP; liver
Chick definitive endoderm is an important source of signals that pattern the early embryo forming a central structure around which the body plan is constructed. Although the origin of definitive endoderm has been mapped in the chick, arising principally from rostral streak at elongating streak stages, it is not known when this layer first becomes fully committed to its germ layer fate, an important issue to resolve in light of its critical role in subsequent patterning of the early embryo.
Through gene expression screening of chick gastrula, we identified molecular markers of definitive endoderm restricted to rostral (Sox17) and caudal (Gata5/6) regions, suggesting that at least two subpopulations of definitive endodermal cells exist during ingression. We show (1) that presumptive mesoderm cells migrate to the middle layer and remain mesenchymal when transplanted to rostral primitive streak, and prospective endoderm cells enter the lower layer and become epithelial when transplanted to caudal primitive streak; and (2) that presumptive endoderm cells and mesoderm cells lose normal gene expression (Sox17 and Wnt8c, respectively) when transplanted outside of their normal position of origin. Moreover, when rostral or caudal primitive streak segments are transplanted into rostral blastoderm isolates (RBIs), both types of transplants express Sox17 4–6 hours later–consistent with their new position, regardless of their presumptive germ layer origin–and prospective mesoderm transplants, which normally express Wnt8c, turn off expression, suggesting that signals within the rostral blastoderm induce endoderm gene expression, and repress mesoderm gene expression, during gastrulation.
Our results demonstrate that germ layer identity is fixed at the time populations of endoderm and mesoderm cells ingress through the primitive streak, whereas their gene expression patterns remain labile. In addition, our results show that inductive and repressive signals are present, and that these signals regulate gene expression of both ingressed endoderm and mesoderm cells. Thus, gastrula cells display elements of both pre-patterning and plasticity, with endoderm the first germ layer becoming committed to its fate during early gastrulation stages.
Organogenesis requires expansion of the embryonic vascular plexus that migrates into developing organs through a process called angiogenesis. Mesodermal progenitors are thought to derive endothelial cells (ECs) that contribute to both embryonic vasculogenesis and the subsequent organ angiogenesis. Here, we demonstrate that during development of the liver, which is an endoderm derivative, a subset of ECs is generated from FOXA2+ endoderm-derived fetal hepatoblast progenitor cells expressing KDR (VEGFR2/FLK-1). Using human and mouse embryonic stem cell models, we demonstrate that KDR+FOXA2+ endoderm cells developing in hepatic differentiation cultures generate functional ECs. This introduces the concept that ECs originate not exclusively from mesoderm but also from endoderm, supported in Foxa2 lineage-tracing mouse embryos by the identification of FOXA2+ cell-derived CD31+ ECs that integrate the vascular network of developing fetal livers.
•Functional ECs are generated from human ESC-derived KDR+ endoderm cells•Functional ECs are generated from mouse ESC-derived KDR+FOXA2+ endoderm cells•FOXA2+ endoderm cells contribute in vivo to a subset of ECs in the mouse fetal liver
In this article, Gouon-Evans and colleagues show that endoderm generates a subset of endothelial cells in the developing liver. The endoderm-derived endothelial cells develop from a hepatic progenitor expressing the VEGF receptor (KDR) in mouse and human embryonic stem cell hepatic differentiation cultures. These bipotential progenitors were confirmed in the developing liver using endoderm lineage-tracing mouse models.
Wnt/β-catenin signaling plays multiple roles in liver development including hepatoblast proliferation and differentiation, hepatocyte differentiation, and liver zonation. A positive role for Wnt/β-catenin signaling in liver specification was recently identified in zebrafish; however, its underlying cellular mechanisms are unknown. Here, we present two cellular mechanisms by which Wnt/β-catenin signaling regulates liver specification. First, using lineage tracing we show that ectopic hepatoblasts, which form in the endoderm posterior to the liver upon activation of Wnt/β-catenin signaling, are derived from the direct conversion of non-hepatic endodermal cells, but not from the posterior migration of hepatoblasts. We found that endodermal cells at the 4–6th somite levels, which normally give rise to the intestinal bulb or intestine, gave rise to hepatoblasts in Wnt8a-overexpressing embryos, and that the distribution of traced endodermal cells in Wnt8a-overexpressing embryos was similar to that in controls. Second, by using an endoderm-restricted cell-transplantation technique and mosaic analysis with transgenic lines that cell-autonomously suppress or activate Wnt/β-catenin signaling upon heat-shock, we show that Wnt/β-catenin signaling acts cell-autonomously in endodermal cells to induce hepatic conversion. Altogether, these data demonstrate that Wnt/β-catenin signaling can induce the fate-change of non-hepatic endodermal cells into a liver fate in a cell-autonomous manner. These findings have potential application to hepatocyte differentiation protocols for the generation of mature hepatocytes from induced pluripotent stem cells, supplying a sufficient amount of hepatocytes for cell-based therapies to treat patients with severe liver diseases.
Hepatoblast; Liver specification; Hepatic conversion; Endoderm; Zebrafish; Fate-change