Definitive Endoderm Formation Research Articles

A sequential EMT-MET mechanism drives the differentiation of human embryonic stem cells towards hepatocytes

Reprogramming has been shown to involve EMT–MET; however, its role in cell differentiation is unclear. We report here that in vitro differentiation of hESCs to hepatic lineage undergoes a sequential EMT–MET with an obligatory intermediate mesenchymal phase. Gene expression analysis reveals that Activin A-induced formation of definitive endoderm (DE) accompanies a synchronous EMT mediated by autocrine TGFβ signalling followed by a MET process. Pharmacological inhibition of TGFβ signalling blocks the EMT as well as DE formation. We then identify SNAI1 as the key EMT transcriptional factor required for the specification of DE. Genetic ablation of SNAI1 in hESCs does not affect the maintenance of pluripotency or neural differentiation, but completely disrupts the formation of DE. These results reveal a critical mesenchymal phase during the acquisition of DE, highlighting a role for sequential EMT–METs in both differentiation and reprogramming.

Genome Editing in hPSCs Reveals GATA6 Haploinsufficiency and a Genetic Interaction with GATA4 in Human Pancreatic Development

Human disease phenotypes associated with haploinsufficient gene requirements are often not recapitulated well in animal models. Here, we have investigated the association between human GATA6 haploinsufficiency and a wide range of clinical phenotypes that include neonatal and adult-onset diabetes using CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9-mediated genome editing coupled with human pluripotent stem cell (hPSC) directed differentiation. We found that loss of one GATA6 allele specifically affects the differentiation of human pancreatic progenitors from the early PDX1+ stage to the more mature PDX1+NKX6.1+ stage, leading to impaired formation of glucose-responsive β-like cells. In addition to this GATA6 haploinsufficiency, we also identified dosage-sensitive requirements for GATA6 and GATA4 in the formation of both definitive endoderm and pancreatic progenitor cells. Our work expands the application of hPSCs from studying the impact of individual gene loci to investigation of multigenic human traits, and it establishes an approach for identifying genetic modifiers of human disease.

Generation of Gastrointestinal Organoids from Human Pluripotent Stem Cells.

Over the past several decades, developmental biologists have discovered fundamental mechanisms by which organs form in developing embryos. With this information it is now possible to generate human "organoids" by the stepwise differentiation of human pluripotent stem cells using a process that recapitulates organ development. For the gastrointestinal tract, one of the first key steps is the formation of definitive endoderm and mesoderm, a process that relies on the TGFb molecule Nodal. Endoderm is then patterned along the anterior-posterior axis, with anterior endoderm forming the foregut and posterior endoderm forming the mid and hindgut. A-P patterning of the endoderm is accomplished by the combined activities of Wnt, BMP, and FGF. High Wnt and BMP promote a posterior fate, whereas repressing these pathways promotes an anterior endoderm fate. The stomach derives from the posterior foregut and retinoic acid signaling is required for promoting a posterior foregut fate. The small and large intestine derive from the mid and hindgut, respectively.These stages of gastrointestinal development can be precisely manipulated through the temporal activation and repression of the pathways mentioned above. For example, stimulation of the Nodal pathway with the mimetic Activin A, another TGF-β superfamily member, can trigger the differentiation of pluripotent stem cells into definitive endoderm (D'Amour et al., Nat Biotechnol 23:1534-1541, 2005). Exposure of definitive endoderm to high levels of Wnt and FGF promotes the formation of posterior endoderm and mid/hindgut tissue that expresses CDX2. Mid-hindgut spheroids that are cultured in a three-dimensional matrix form human intestinal organoids (HIOs) that are small intestinal in nature Spence et al., Nature 2011. In contrast, activation of FGF and Wnt in the presence of the BMP inhibitor Noggin promotes the formation of anterior endoderm and foregut tissues that express SOX2. These SOX2-expressing foregut spheroids can be further patterned into posterior foregut by addition of retinoic acid. Once formed, these posterior foregut spheroids can be grown in three-dimensional human gastric organoids (HGOs) that have all of the cell types of antral part of the stomach (Mc Cracken et al. 2014).Here, we describe the detailed methods for generating stomach/human gastric organoids (HGOs) and human intestinal organoids (HIOs) from human pluripotent stem cells. We first present a method for generating definitive endoderm from pluripotent stem cells followed by differentiation of definitive endoderm into either posterior foregut spheroids or mid-hindgut spheroids. We then describe how three-dimensional culturing of these spheroids results in the formation of HGOs and HIOs, respectively.

Functional characterisation of cis-regulatory elements governing dynamic Eomes expression in the early mouse embryo.

The T-box transcription factor (TF) Eomes is a key regulator of cell fate decisions during early mouse development. The cis-acting regulatory elements that direct expression in the anterior visceral endoderm (AVE), primitive streak (PS) and definitive endoderm (DE) have yet to be defined. Here, we identified three gene-proximal enhancer-like sequences (PSE_a, PSE_b and VPE) that faithfully activate tissue-specific expression in transgenic embryos. However, targeted deletion experiments demonstrate that PSE_a and PSE_b are dispensable, and only VPE is required for optimal Eomes expression in vivo. Embryos lacking this enhancer display variably penetrant defects in anterior-posterior axis orientation and DE formation. Chromosome conformation capture experiments reveal VPE-promoter interactions in embryonic stem cells (ESCs), prior to gene activation. The locus resides in a large (500 kb) pre-formed compartment in ESCs and activation during DE differentiation occurs in the absence of 3D structural changes. ATAC-seq analysis reveals that VPE, PSE_a and four additional putative enhancers display increased chromatin accessibility in DE that is associated with Smad2/3 binding coincident with transcriptional activation. By contrast, activation of the Eomes target genes Foxa2 and Lhx1 is associated with higher order chromatin reorganisation. Thus, diverse regulatory mechanisms govern activation of lineage specifying TFs during early development.

GATA6 is essential for endoderm formation from human pluripotent stem cells.

ABSTRACTProtocols have been established that direct differentiation of human pluripotent stem cells into a variety of cell types, including the endoderm and its derivatives. This model of differentiation has been useful for investigating the molecular mechanisms that guide human developmental processes. Using a directed differentiation protocol combined with shRNA depletion we sought to understand the role of GATA6 in regulating the earliest switch from pluripotency to definitive endoderm. We reveal that GATA6 depletion during endoderm formation results in apoptosis of nascent endoderm cells, concomitant with a loss of endoderm gene expression. We show by chromatin immunoprecipitation followed by DNA sequencing that GATA6 directly binds to several genes encoding transcription factors that are necessary for endoderm differentiation. Our data support the view that GATA6 is a central regulator of the formation of human definitive endoderm from pluripotent stem cells by directly controlling endoderm gene expression.

The molecular mechanisms and their omic studies in the endoderm-lineage differentiation of embryonic stem cells

The embryonic stem cells (ESC) can be differentiated into three germ layers, known as endoderm, mesoderm and ectoderm. Endoderm, which generates terminally differentiated cells, lays the foundation for generating the respiratory and digestive systems as well as the functional organs such as liver and pancreas. The differentiation of ESC into endoderm includes four main stages: the differentiation into epiblast, the formation of primitive streak, the separation of endoderm and mesoderm, the eventual formation of definitive endoderm (DE). The molecular mechanisms of endoderm-lineage differentiation of ESC are summarized in this review. We mainly focus on the key signaling pathways, transcription factors, epigenetic regulation mechanisms in the four processes. The progress of the multi-omic studies of these mechanisms are highlighted. This review may provide important referenced information for the researchers in this field.

Generation of functional hepatocyte-like cells from human deciduous periodontal ligament stem cells.

Human deciduous periodontal ligament stem cells have been introduced for as an easily accessible source of stem cells from dental origin. Although recent studies have revealed the ability of these stem cells in multipotential attribute, their efficiency of hepatic lineage differentiation has not been addressed so far. The aim of this study is to investigate hepatic lineage fate competence of periodontal ligament stem cells through direct media induction. Differentiation of periodontal ligament stem cells into hepatocyte-like cells was conducted by the exposure of two phase media induction. First phase was performed in the presence of hepatocyte growth factors to induce a definitive endoderm formation. In the subsequent phase, the cells were treated with oncostatin M and dexamethosone followed by insulin and transferrin to generate hepatocyte-like cells. Hepatic-related characters of the generated hepatocyte-like cells were determined at both mRNA and protein level followed by functional assays. Foremost changes observed in the generation of hepatocyte-like cells were the morphological features in which these cells were transformed from fibroblastic shape to polygonal shape. Temporal expression of hepatic markers ranging from early endodermal up to late markers were detected in the hepatocyte-like cells. Crucial hepatic markers such as glycogen storage, albumin, and urea secretion were also shown. These findings exhibited the ability of periodontal ligament stem cells of dental origin to be directed into hepatic lineage fate. These cells can be regarded as an alternative autologous source in the usage of stem cell-based treatment for liver diseases.

Efficient generation of functional pancreatic β-cells from human induced pluripotent stem cells.

Insulin-secreting cells have been generated from human embryonic or induced pluripotent stem cells (iPSCs) by mimicking developmental processes. However, these cells do not always secrete glucose-responsive insulin, one of the most important characteristics of pancreatic β-cells. We focused on the importance of endodermal differentiation from human iPSCs in order to obtain functional pancreatic β-cells. A six-stage protocol was established for the differentiation of human iPSCs to pancreatic β-cells using defined culture media without feeders or serum. The effects of CHIR99021, a selective glycogen synthase kinase-3β inhibitor, were examined in the presence of fibroblast growth factor 2, activin, and bone morphogenetic protein 4 (FAB) during definitive endodermal induction by immunostaining for SRY (sex determining region Y)-box 17 (SOX17) and Forkhead box protein A2 (FOXA2). Insulin secretion was compared between the last stage of monolayer culture and spheroid culture conditions. Cultured cells were transplanted under kidney capsules of streptozotocin-diabetic non-obese diabetic-severe combined immunodeficiency mice, and blood glucose levels were measured once a week. Immunohistochemical analyses were performed 4 and 12 weeks after transplantation. Addition of CHIR99021 (3 μmol/L) in the presence of FAB for 2 days improved endodermal cell viability, maintaining the high SOX17-positive rate. Spheroid formation after the endocrine progenitor stage showed more efficient insulin secretion than did monolayer culture. After cell transplantation, diabetic mice had lower blood glucose levels, and islet-like structures were detected in vivo. Functional pancreatic β-cells were generated from human iPSCs. Induction of definitive endoderm and spheroid formation may be key steps for producing these cells.

Biphasic modulation of Wnt signaling supports efficient foregut endoderm formation from human pluripotent stem cells.

Pluripotent stem cells (embryonic stem cells and induced pluripotent stem cells) are of great promise in regenerative medicine, including molecular studies of disease mechanisms, if the affected cell type can be authentically generated during in vitro differentiation. Most existing protocols aim to mimic embryonic development steps by the supplementation of specific cytokines and small molecules, but the involved signaling pathways need further exploration. In this study, we investigated enhanced initial activation of Wnt signaling for definitive endoderm formation and subsequent rapid shutdown of Wnt signaling for proper foregut endoderm specification using 3 μM CHIR99021 and 0.5 μg/mL of secreted frizzled-related protein 5 (sFRP-5) for biphasic modulation of the Wnt pathway. The definitive endoderm and foregut endoderm differentiation capabilities of Wnt pathway-modulated cells were determined based on the expression levels of the endodermal transcription factors SOX17 and FOXA2 and those of the transcription activator GATA4 and the α-fetoprotein (AFP) gene, respectively. Furthermore, the resulting biphasic Wnt pathway modulation was investigated at the protein level by analyzing phosphorylation of glycogen synthase kinase 3 beta (GSK3β) and β-catenin. Finally, Wnt target gene expression was determined using an improved lentiviral reporter construct that enabled robust T-cell transcription factor 4 (TCF4)/lymphoid enhancer-binding factor 1 (LEF1)-mediated luciferase expression in differentiating pluripotent stem cells. In conclusion, we demonstrated robust, homogeneous, and efficient derivation of foregut endodermal cells by inducing a biphasic modulation of the Wnt signaling pathway.

Signaling and Gene Regulatory Networks Governing Definitive Endoderm Derivation From Pluripotent Stem Cells.

The generation of definitive endoderm (DE) from pluripotent stem cells (PSCs) is a fundamental stage in the formation of highly organized visceral organs, such as the liver and pancreas. Currently, there is a need for a comprehensive study that illustrates the involvement of different signaling pathways and their interactions in the derivation of DE cells from PSCs. This study aimed to identify signaling pathways that have the greatest influence on DE formation using analyses of transcriptional profiles, protein-protein interactions, protein-DNA interactions, and protein localization data. Using this approach, signaling networks involved in DE formation were constructed using systems biology and data mining tools, and the validity of the predicted networks was confirmed experimentally by measuring the mRNA levels of hub genes in several PSCs-derived DE cell lines. Based on our analyses, seven signaling pathways, including the BMP, ERK1-ERK2, FGF, TGF-beta, MAPK, Wnt, and PIP signaling pathways and their interactions, were found to play a role in the derivation of DE cells from PSCs. Lastly, the core gene regulatory network governing this differentiation process was constructed. The results of this study could improve our understanding surrounding the efficient generation of DE cells for the regeneration of visceral organs. J. Cell. Physiol. 231: 1994-2006, 2016. © 2016 Wiley Periodicals, Inc.

Convergence of cMyc and β-catenin on Tcf7l1 enables endoderm specification.

The molecular machinery that directs formation of definitive endoderm from pluripotent stem cells is not well understood. Wnt/β-catenin and Nodal signalling have been implicated, but the requirements for lineage specification remain incompletely defined. Here, we demonstrate a potent effect of inhibiting glycogen synthase kinase 3 (GSK3) on definitive endoderm production. We find that downstream of GSK3 inhibition, elevated cMyc and β-catenin act in parallel to reduce transcription and DNA binding, respectively, of the transcriptional repressor Tcf7l1. Tcf7l1 represses FoxA2, a pioneer factor for endoderm specification. Deletion of Tcf7l1 is sufficient to allow upregulation of FoxA2 in the presence of Activin. In wild-type cells, cMyc contributes by reducing Tcf7l1 mRNA, while β-catenin acts on Tcf7l1 protein. GSK3 inhibition is further required for consolidation of endodermal fate via upregulation of Sox17, highlighting sequential roles for Wnt signalling. The identification of a cMyc/β-catenin-Tcf7l1-FoxA2 axis reveals a de-repression mechanism underlying endoderm induction that may be recapitulated in other developmental and patho-logical contexts.

Significance of microRNAs for the differentiation of human embryonic stem cells into definitive endoderm and mesoderm

Research Question: Differentiation of pluripotent stem cells into endoderm-derived cells, like insulin-producing cells, requires initially the formation of definitive endoderm (DE). MicroRNAs are important post-transcriptional regulators of the gene expression determining cell fates. Therefore, this study analyzed the microRNA expression profiles of purified DE, mesodermal cells, randomly differentiated cells, and undifferentiated ESC to identify important early cell fate regulators.

DMSO efficiently down regulates pluripotency genes in human embryonic stem cells during definitive endoderm derivation and increases the proficiency of hepatic differentiation.

BackgroundDefinitive endoderm (DE) is one of the three germ layers which during in vivo vertebrate development gives rise to a variety of organs including liver, lungs, thyroid and pancreas; consequently efficient in vitro initiation of stem cell differentiation to DE cells is a prerequisite for successful cellular specification to subsequent DE-derived cell types [1, 2]. In this study we present a novel approach to rapidly and efficiently down regulate pluripotency genes during initiation of differentiation to DE cells by addition of dimethyl sulfoxide (DMSO) to Activin A-based culture medium and report its effects on the downstream differentiation to hepatocyte-like cells.Materials and MethodsHuman embryonic stem cells (hESC) were differentiated to DE using standard methods in medium supplemented with 100ng/ml of Activin A and compared to cultures where DE specification was additionally enhanced with different concentrations of DMSO. DE cells were subsequently primed to generate hepatic-like cells to investigate whether the addition of DMSO during formation of DE improved subsequent expression of hepatic markers. A combination of flow cytometry, real-time quantitative reverse PCR and immunofluorescence was applied throughout the differentiation process to monitor expression of pluripotency (POUF5/OCT4 & NANOG), definitive endoderm (SOX17, CXCR4 & GATA4) and hepatic (AFP & ALB) genes to generate differentiation stage-specific signatures.ResultsAddition of DMSO to the Activin A-based medium during DE specification resulted in rapid down regulation of the pluripotency genes OCT4 and NANOG, accompanied by an increase expression of the DE genes SOX17, CXCR4 and GATA4. Importantly, the expression level of ALB in DMSO-treated cells was also higher than in cells which were differentiated to the DE stage via standard Activin A treatment.

Recent developments in β-cell differentiation of pluripotent stem cells induced by small and large molecules.

Human pluripotent stem cells, including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), hold promise as novel therapeutic tools for diabetes treatment because of their self-renewal capacity and ability to differentiate into beta (β)-cells. Small and large molecules play important roles in each stage of β-cell differentiation from both hESCs and hiPSCs. The small and large molecules that are described in this review have significantly advanced efforts to cure diabetic disease. Lately, effective protocols have been implemented to induce hESCs and human mesenchymal stem cells (hMSCs) to differentiate into functional β-cells. Several small molecules, proteins, and growth factors promote pancreatic differentiation from hESCs and hMSCs. These small molecules (e.g., cyclopamine, wortmannin, retinoic acid, and sodium butyrate) and large molecules (e.g. activin A, betacellulin, bone morphogentic protein (BMP4), epidermal growth factor (EGF), fibroblast growth factor (FGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), noggin, transforming growth factor (TGF-α), and WNT3A) are thought to contribute from the initial stages of definitive endoderm formation to the final stages of maturation of functional endocrine cells. We discuss the importance of such small and large molecules in uniquely optimized protocols of β-cell differentiation from stem cells. A global understanding of various small and large molecules and their functions will help to establish an efficient protocol for β-cell differentiation.

Differential response of epiblast stem cells to Nodal and Activin signalling: a paradigm of early endoderm development in the embryo.

Mouse epiblast stem cells (EpiSCs) display temporal differences in the upregulation of Mixl1 expression during the initial steps of in vitro differentiation, which can be correlated with their propensity for endoderm differentiation. EpiSCs that upregulated Mixl1 rapidly during differentiation responded robustly to both Activin A and Nodal in generating foregut endoderm and precursors of pancreatic and hepatic tissues. By contrast, EpiSCs that delayed Mixl1 upregulation responded less effectively to Nodal and showed an overall suboptimal outcome of directed differentiation. The enhancement in endoderm potency in Mixl1-early cells may be accounted for by a rapid exit from the progenitor state and the efficient response to the induction of differentiation by Nodal. EpiSCs that readily differentiate into the endoderm cells are marked by a distinctive expression fingerprint of transforming growth factor (TGF)-β signalling pathway genes and genes related to the endoderm lineage. Nodal appears to elicit responses that are associated with transition to a mesenchymal phenotype, whereas Activin A promotes gene expression associated with maintenance of an epithelial phenotype. We postulate that the formation of definitive endoderm (DE) in embryoid bodies follows a similar process to germ layer formation from the epiblast, requiring an initial de-epithelialization event and subsequent re-epithelialization. Our results show that priming EpiSCs with the appropriate form of TGF-β signalling at the formative phase of endoderm differentiation impacts on the further progression into mature DE-derived lineages, and that this is influenced by the initial characteristics of the cell population. Our study also highlights that Activin A, which is commonly used as an in vitro surrogate for Nodal in differentiation protocols, does not elicit the same downstream effects as Nodal, and therefore may not effectively mimic events that take place in the mouse embryo.

Hypoxia enhances differentiation of mouse embryonic stem cells into definitive endoderm and distal lung cells.

We investigated the effects of hypoxia on spontaneous (SP)- and activin A (AA)-induced definitive endoderm (DE) differentiation of mouse embryonic stem cells (mESCs) and their subsequent differentiation into distal pulmonary epithelial cells. SP differentiation for 6 days of mESCs toward endoderm at hypoxia of 1% O2, but not at 3% or 21% (normoxia), increased the expression of Sox17 and Foxa2 by 31- and 63-fold above maintenance culture, respectively. Treatment of mESCs with 20 ng/mL AA for 6 days under hypoxia further increased the expression of DE marker genes Sox17, Foxa2, and Cxcr4 by 501-, 1,483-, and 126-fold above maintenance cultures, respectively. Transient exposure to hypoxia, as short as 24 h, was sufficient to enhance AA-induced endoderm formation. The involvement of hypoxia-inducible factor (HIF)-1α and reactive oxygen species (ROS) in the AA-induced endoderm enrichment was assessed using HIF-1α(-/-) mESCs and the ROS scavenger N-acetylcysteine (NAC). Under SP conditions, HIF-1α(-/-) mESCs failed to increase the expression of endodermal marker genes but rather shifted toward ectoderm. Hypoxia induced only a marginal potentiation of AA-induced endoderm differentiation in HIF-1α(-/-) mESCs. Treatment of mESCs with AA and NAC led to a dose-dependent decrease in Sox17 and Foxa2 expression. In addition, the duration of exposure to hypoxia in the course of a recently reported lung differentiation protocol resulted in differentially enhanced expression of distal lung epithelial cell marker genes aquaporin 5 (Aqp5), surfactant protein C (Sftpc), and secretoglobin 1a1 (Scgb1a1) for alveolar epithelium type I, type II, and club cells, respectively. Our study is the first to show the effects of in vitro hypoxia on efficient formation of DE and lung lineages. We suggest that the extent of hypoxia and careful timing may be important components of in vitro differentiation bioprocesses for the differential generation of distal lung epithelial cells from pluripotent progenitors.

SRNA-seq analysis of human embryonic stem cells and definitive endoderm reveals differentially expressed microRNAs and novel IsomiRs with distinct targets.

MicroRNAs (miRNAs) are noncoding, regulatory RNAs expressed dynamically during differentiation of human embryonic stem cells (hESCs) into defined lineages. Mapping developmental expression of miRNAs during transition from pluripotency to definitive endoderm (DE) should help to elucidate the mechanisms underlying lineage specification and ultimately enhance differentiation protocols. In this report, next generation sequencing was used to build upon our previous analysis of miRNA expression in human hESCs and DE. From millions of sequencing reads, 747 and 734 annotated miRNAs were identified in pluripotent and DE cells, respectively, including 77 differentially expressed miRNAs. Among these, four of the top five upregulated miRNAs were previously undetected in DE. Furthermore, the stem-loop for miR-302a, an important miRNA for both hESCs self-renewal and endoderm specification, produced several highly expressed miRNA species (isomiRs). Overall, isomiRs represented >10% of sequencing reads in >40% of all detected stem-loop arms, suggesting that the impact of these abundant miRNA species may have been overlooked in previous studies. Because of their relative abundance, the role of differential isomiR targeting was studied using the miR-302 cluster as a model system. A miRNA mimetic for miR-302a-5p, but not miR-302a-5p(+3), decreased expression of orthodenticle homeobox 2 (OTX2). Conversely, isomiR 302a-5p(+3) selectively decreased expression of tuberous sclerosis protein 1, but not OTX2, indicating nonoverlapping specificity of miRNA processing variants. Taken together, our characterization of miRNA expression, which includes novel miRNAs and isomiRs, helps establish a foundation for understanding the role of miRNAs in DE formation and selective targeting by isomiRs.

Comparable Generation of Activin-Induced Definitive Endoderm via Additive Wnt or BMP Signaling in Absence of Serum

SummaryThere is considerable interest in differentiating human pluripotent stem cells (hPSCs) into definitive endoderm (DE) and pancreatic cells for in vitro disease modeling and cell replacement therapy. Numerous protocols use fetal bovine serum, which contains poorly defined factors to induce DE formation. Here, we compared Wnt and BMP in their ability to cooperate with Activin signaling to promote DE formation in a chemically defined medium. Varying concentrations of WNT3A, glycogen synthase kinase (GSK)-3 inhibitors CHIR99021 and 6-bromoindirubin-3′-oxime (BIO), and BMP4 could independently co-operate with Activin to effectively induce DE formation even in the absence of serum. Overall, CHIR99021 is favored due to its cost effectiveness. Surprisingly, WNT3A was ineffective in suppressing E-CADHERIN/CDH1 and pluripotency factor gene expression unlike GSK-3 inhibitors or BMP4. Our findings indicate that both Wnt and BMP effectively synergize with Activin signaling to generate DE from hPSCs, although WNT3A requires additional factors to suppress the pluripotency program inherent in hPSCs.

The formation of proximal and distal definitive endoderm populations in culture requires p38 MAPK activity

Endoderm formation in the mammal is a complex process with two lineages forming during the first weeks of development, the primitive (or extraembryonic) endoderm, which is specified in the blastocyst, and the definitive endoderm that forms later, at gastrulation, as one of the germ layers of the embryo proper. Fate mapping evidence suggests that the definitive endoderm arises as two waves, which potentially reflect two distinct cell populations. Early primitive ectoderm-like (EPL) cell differentiation has been used successfully to identify and characterise mechanisms regulating molecular gastrulation and lineage choice during differentiation. The roles of the p38 MAPK family in the formation of definitive endoderm were investigated using EPL cells and chemical inhibitors of p38 MAPK activity. These approaches define a role for p38 MAPK activity in the formation of the primitive streak and a second role in the formation of the definitive endoderm. Characterisation of the definitive endoderm populations formed from EPL cells demonstrates the formation of two distinct populations, defined by gene expression and ontogeny, that were analogous to the proximal and distal definitive endoderm populations of the embryo. Formation of the proximal definitive endoderm was found to require p38 MAPK activity and is correlated with molecular gastrulation, defined by the expression of brachyury (T). Distal definitive endoderm formation also requires p38 MAPK activity but can form when T expression is inhibited. Understanding lineage complexity will be a prerequisite for the generation of endoderm derivatives for commercial and clinical use.

Endoderm complexity in the mouse gastrula is revealed through the expression of spink3.

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.