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In drug discovery, there is an increasing demand for more physiological in vitro models that recapitulate the disease situation in patients. Human induced pluripotent stem (hiPS) cell-derived model cells could serve this purpose. To date, several directed differentiation approaches have been described to generate definitive endoderm (DE) from hiPS cells, but protocols suitable for drug development and high-throughput screening (HTS) have not been reported yet. In this work, a large-scale expansion of hiPS cells for high-throughput adaption is presented and an optimized stepwise differentiation of hiPS cells into DE cells is described. The produced DE cells were demonstrated to express classical DE markers on the gene expression and protein level. The here described DE cells are multipotent progenitors and act as starting points for a broad spectrum of endodermal model cells in HTS and other areas of drug discovery.

Summary Efficient stem cell differentiation into pancreatic islet cells is of critical importance for the development of cell replacement therapies for diabetes. Here, we identify the expression pattern of connexin 43 (Cx43), a gap junction (GJ) channel protein, in human embryonic stem cell (hESC)-derived definitive endoderm (DE) and primitive gut tube cells, representing early lineages for posterior foregut (PF), pancreatic progenitors (PP), pancreatic endocrine progenitors (PE), and islet cells. As the function of GJ channels is dependent on their gating status, we tested the impact of supplementing hESC-derived PP cell cultures with AAP10, a peptide that promotes Cx43 GJ channel opening. We found that this treatment promotes the expression of DE markers FoxA2 and Sox17, leads to a more efficient derivation of DE, and improves the yield of PF, PP, and PE cells. These results demonstrate a functional involvement of GJ channels in the differentiation of embryonic stem cells into pancreatic cell lineages., Graphical Abstract, Highlights • Cx43 is upregulated during hESC differentiation into definitive endoderm (DE) • siRNA-mediated knockdown of Cx43 prevents hESC differentiation into DE cells • AAP10-mediated gating of Cx43 gap junctions promotes the expression of FoxA2 and Sox17 • Cx43 gap junction communication supports DE and pancreatic progenitors development, Cell Biology; Stem Cells Research; Specialized Functions of Cells

There is substantial interest in the evolutionary forces that shaped the regulatory framework that is established in early human development. Progress in this area has been slow because it is difficult to obtain relevant biological samples. Inducible pluripotent stem cells (iPSCs) provide the ability to establish in vitro models of early human and non-human primate developmental stages. Using matched iPSC panels from humans and chimpanzees, we comparatively characterized gene regulatory changes through a four-day timecourse differentiation of iPSCs (day 0) into primary streak (day 1), endoderm progenitors (day 2), and definitive endoderm (day 3). As might be expected, we found that differentiation stage is the major driver of variation in gene expression levels, followed by species. We identified thousands of differentially expressed genes between humans and chimpanzees in each differentiation stage. Yet, when we considered gene-specific dynamic regulatory trajectories throughout the timecourse, we found that 75‥ of genes, including nearly all known endoderm developmental markers, have similar trajectories in the two species. Interestingly, we observed a marked reduction of both intra-and inter-species variation in gene expression levels in primitive streak samples compared to the iPSCs, with a recovery of regulatory variation in endoderm progenitors. The reduction of variation in gene expression levels at a specific developmental stage, paired with overall high degree of conservation of temporal gene regulation, is consistent with the dynamics of developmental canalization. Overall, we conclude that endoderm development in iPSC-based models are highly conserved and canalized between humans and our closest evolutionary relative.

The introduction of human induced pluripotent stem cells (hiPSCs) for the generation of various cell types allows the development of more realistic and patient-specific tissue models. A major advantage is the possibility to obtain multiple cell types with the same genetic background from the same hiPSC line. The aim of the present study was to develop alveolar epithelial type 2 (AT2) cells from hiPSCs in order to develop a model of alveolar wound repair. Therefore, hiPSCs were gradually differentiated towards an alveolar and endothelial fate by closely controlling the BMP, FGF, WNT, RA and TGF-β signalling pathways. First, definitive endoderm was induced over a period of 4 days, followed by the formation of anterior foregut endoderm and subsequently ventralized over a period of 12 days. Following ventralization, alveolar progenitors were generated during 8 days and finally the cells were exposed to dexamethasone and KGF for 4-6 weeks to induce expression of alveolar epithelial type 2 markers (surfactant proteins and LP-CAT 1) as well as lamellar bodies in about 20% of the cells. Using an EpCAM isolation we managed to setup a long-term culture of induced ATII cells at the air liquid interface (ALI). Using this setup we were able to visualize and monitor alveolar repair over the course of 2 weeks following mechanical wounding of the cultures. In a next phase of our research, we will compare wound repair of primary and hiPSC-derived ATII cells.

Embryonic stem cells can be incorporated into the developing embryo and its germ line, but, when cultured alone, their ability to generate embryonic structures is restricted. They can interact with trophoblast stem cells to generate structures that break symmetry and specify mesoderm, but their development is limited as the epithelial-mesenchymal transition of gastrulation cannot occur. Here, we describe a system that allows assembly of mouse embryonic, trophoblast and extra-embryonic endoderm stem cells into structures that acquire the embryo's architecture with all distinct embryonic and extra-embryonic compartments. Strikingly, such embryo-like structures develop to undertake the epithelial-mesenchymal transition, leading to mesoderm and then definitive endoderm specification. Spatial transcriptomic analyses demonstrate that these morphological transformations are underpinned by gene expression patterns characteristic of gastrulating embryos. This demonstrates the remarkable ability of three stem cell types to self-assemble in vitro into gastrulating embryo-like structures undertaking spatio-temporal events of the gastrulating mammalian embryo.

The development of complex in vitro hepatic systems and artificial liver devices has been hampered by the lack of reliable sources for relevant cell types, such as hepatic stellate cells (HSCs). Here we report efficient differentiation of human pluripotent stem cells into HSC-like cells (iPSC-HSCs). iPSC-HSCs closely resemble primary human HSCs at the transcriptional, cellular, and functional levels and possess a gene expression profile intermediate between that of quiescent and activated HSCs. Functional analyses revealed that iPSC-HSCs accumulate retinyl esters in lipid droplets and are activated in response to mediators of wound healing, similar to their in vivo counterparts. When maintained as 3D spheroids with HepaRG hepatocytes, iPSC-HSCs exhibit a quiescent phenotype but mount a fibrogenic response and secrete pro-collagen in response to known stimuli and hepatocyte toxicity. Thus, this protocol provides a robust in vitro system for studying HSC development, modeling liver fibrosis, and drug toxicity screening. ispartof: CELL STEM CELL vol:23 issue:1 pages:101-+ ispartof: location:United States status: published

Hepatocyte-like cells differentiated from human iPS cells are expected to be utilized in pharmaceutical research and regenerative medicine. Recently, various culture methods for human iPS cell maintenance have been developed. However, it is not well known whether human iPS cell maintenance method affects hepatic differentiation potency. In this study, we cultured human iPS cells using four maintenance methods: ReproStem medium with feeder cells (mouse embryonic fibroblasts), AK02N medium with iMatrix-511 (E8 fragments of laminin511), Essential 8 medium with Vitronectin N (N-terminal domain of vitronectin), TeSR-E8 medium with Vitronectin XF (xeno-free vitronectin). Then, these human iPS cells were differentiated into the hepatocyte-like cells. Interestingly, the gene expression levels of definitive endoderm markers in the definitive endoderm cells generated from human iPS cells cultured with ReproStem or TeSR-E8 medium were higher than those in other groups. The gene expression level of foregut marker, HHEX, in the definitive endoderm cells generated from human iPS cells cultured with ReproStem medium was higher than that in other groups. Consistently, the expression levels of hepatocyte markers, albumin and urea secretion capacity, and CYP3A4 activity in the hepatocyte-like cells generated from human iPS cells cultured with ReproStem medium were higher than those in the other groups. Our data indicated that the most suitable human iPS cell maintenance method for efficient hepatic differentiation was the on-feeder method which uses mouse embryonic fibroblasts, but not feeder-free methods. In conclusion, human iPS cell maintenance method largely affects hepatic differentiation potency.

Induced pluripotent stem cells (iPSCs) and human embryonic stem cells (hESCs) differentiated into hepatocyte-like cells (HLCs) provide a defined and renewable source of cells for drug screening, toxicology and regenerative medicine. We previously reprogrammed human fetal foreskin fibroblast cells (HFF1) into iPSCs employing an episomal plasmid-based integration-free approach, this iPSC-line and the hESC lines H1 and H9 were used to model hepatogenesis in vitro. Biochemical characterisation confirmed glycogen storage, ICG uptake and release, urea and bile acid production, as well as CYP3A4 activity. Microarray-based transcriptome analyses was carried out using RNA isolated from the undifferentiated pluripotent stem cells and subsequent differentiation stages- definitive endoderm (DE) hepatic endoderm (HE) and HLCs. K-means identified 100 distinct clusters, for example, POU5F1/OCT4 marking the undifferentiated stage, SOX17 the DE stage, HNF4α the HE stage, and ALB specific to HLCs, fetal liver and primary human hepatocytes (PHH). This data descriptor describes these datasets which should be useful for gaining new insights into the molecular basis of hepatogenesis and associated gene regulatory networks.

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.

Transcription factors are tools repetitively used by the embryo to generate a variety of lineages. Hence, their context of activation is an important determinant of their ability to specifically trigger certain cell fates, but not others. The context is also consequential when considering directing differentiation of embryonic stem cells (ESCs). In this study, we sought to assess the context of pancreatic transcription factor 1a (PTF1a) activation in reference to its propancreatic effects in mouse ESCs (mESCs). We hypothesized that an enriched endodermal population would respond to PTF1a and trigger the pancreatic program more effectively than a spontaneously differentiated population. Using anin vitromodel of pancreas development that we recently established, we found that inducing PTF1a in highly enriched definitive endoderm did not promote pancreatic differentiation but induction in more differentiated endoderm, specifically posterior foregut endoderm, did form pancreatic progenitors. These progenitors never underwent terminal differentiation to endocrine or acinar phenotype. However, a short 3D culture period, prior to PTF1a induction, led to the generation of monohormonal insulin+cells and amylase-expressing cells. Our findings suggest that enriched posterior foregut endoderm is competent to respond to PTF1a’s propancreatic activity; but a 3D culture environment is essential for terminal differentiation of pancreatic progenitors.

Human embryonic stem cells (hESCs) are characterized by both their pluripotency and ability to self-renew, rendering them attractive for treatment of degenerative diseases. The cues necessary to induce hESC differentiation to a desired lineage can be categorized as either chemical or biophysical in nature. While chemical cues are used as primary inducers of differentiation, biophysical cues have been found to augment the process. In this regard, we have designed a novel chitosan nanoparticle augmented encapsulated alginate (CNPEA) platform which can not only augment, but induce differentiation of hESCs into the definitive endoderm (DE) lineage in the absence of specific soluble chemical inducers. These endoderm cells were comparable in phenotype with chemically driven DE cells and remained amenable to further maturation into pancreatic lineages. This study demonstrates the feasibility of carefully designed and tailored nanomaterials inducing differentiation, and moreover demonstrating the possibility of replacing growth factors by material cues.

Summary We demonstrate that the pluripotency gene OCT4 has a role in regulating differentiation via Wnt signaling. OCT4 expression levels in human embryonic stem cells increases transiently during the first 24 hr of in vitro differentiation, with OCT4 occupancy increasing at endoderm regulators such as SOX17 and FOXA2. This increased occupancy correlates with loss of the PRC2 complex and the inhibitory histone mark H3K27me3. Knockdown of OCT4 during differentiation inhibits mesendoderm formation and removal of the H3K27me3 mark from the SOX17 promoter, suggesting that OCT4 acts to induce removal of the PRC2 complex. Furthermore, OCT4 and β-catenin can be co-immunoprecipitated upon differentiation, and Wnt stimulation is required for the enhanced OCT4 occupancy and loss of the PRC2 complex from the SOX17 promoter. In conclusion, our study reveals that OCT4, a master regulator of pluripotency, may also collaborate with Wnt signaling to drive endoderm induction by pre-patterning epigenetic markers on endodermal promoters., Highlights • OCT4 occupancy increases at endoderm genes early in ES cell differentiation • The PRC2 complex is lost from OCT4 sites on endoderm genes during differentiation • OCT4 associates with β-catenin during ES cell differentiation • OCT4 and Wnt are both required for mesendoderm induction, In this article, Gadue and colleagues show that OCT4 in collaboration with WNT signaling acts to modulate epigenetic marks at definitive endoderm genes as little as 24 hr after differentiation initiation, priming them for eventual expression. This study demonstrates that a well-described regulator of pluripotency, OCT4, can also help direct downstream differentiation.

The intestine and colon carries out vital functions, and their lifelong maintenance is of the upmost importance. Research over the past decades has carefully addressed bowel function, how it is maintained and begun to unravel how disorders such as cancer and inflammatory bowel disease form. In contrast, very little is known about the molecular mechanisms that trigger tissue maturation during development. With this review, our aim is to carefully provide a critical appraisal of the literature to give a state-of-the-art view of intestinal development. Starting from definitive endoderm at gastrulation to the emergence of a structure with mature properties, the tissue undergoes complex morphogenetic processes that rely on both biophysical changes and secreted signaling molecules. We will also discuss how new and exciting developments using in vitro models are likely to provide new insights into this process and potential therapeutic strategies for gastrointestinal disorders.

Derivation of definitive endoderm (DE) from human embryonic stem cells (hESCs) can address the needs of regenerative medicine for endoderm-derived organs such as the pancreas and liver. Fibrous substrates which topographically recapitulate native extracellular matrix have been known to promote the stem cell differentiation. However, the optimal fiber diameter remains to be determined for the desired differentiation. Here, we have developed a simple method to precisely fabricate electrospun poly(e-caprolactone) fibers with four distinct average diameters at nano- and microscale levels (200, 500, 800, and 1300 nm). Human ESCs were cultured as clumps or single cells and induced into DE differentiation to determine the optimal topography leading to the promoted differentiation compared with planar culture plates. Gene expression analysis of the DE-induced cells showed significant upregulation of DE-specific genes exclusively on the 200-nm fibers. By Western blot analysis, significant expression of DE-specific proteins was found when hESCs were cultured on the 200 nm substrate as single cells rather than clumps, probably due to more efficient cell–matrix interaction realized by morphological observations of the cell colonies. The results indicated that nanofibrillar substrates, only at ultrathin fiber diameters, provided a better environment for DE differentiation of hESC, which holds great promise in prospective tissue engineering applications. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 3539–3553, 2015.

Porcine embryonic stem cells (ES) are considered attractive preclinical research tools for human liver diseases. Although several studies previously reported generation of porcine ES, none of these studies has described hepatic differentiation from porcine ES. The aim of this study was to generate hepatocytes from porcine ES and analyze their characteristics. We optimized conditions for definitive endoderm induction and developed a 4-step hepatic differentiation protocol. A brief serum-free condition with activin A efficiently induced definitive endoderm differentiation from porcine ES. The porcine ES-derived hepatocyte-like cells highly expressed hepatic markers including albumin and α-fetoprotein, and displayed liver characteristics such as glycogen storage, lipid production, and low-density lipoprotein uptake. For the first time, we describe a highly efficient protocol for hepatic differentiation from porcine ES. Our findings provide valuable information for translational liver research using porcine models, including hepatic regeneration and transplant studies, drug screening, and toxicology.

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.

Dynamic suspension culture of human pluripotent stem cells (hPSCs) in stirred bioreactors provides a valuable scalable culture platform for integrated differentiation toward different lineages for potential research and therapeutic applications. However, current protocols for scalable and integrated differentiation of hPSCs limited due to high cost of growth factors and technical challenges. Here, hPSCs aggregates primed with 6 and 12 μM of CHIR99021 (CHIR), a Wnt agonist, in combination with different concentrations of high cost Activin A (10, 25, 50, 100 ng/mL). We sought to determine the appropriate treatment duration for efficient and cost-effective differentiation protocol for foregut definitive endoderm production in a dynamic suspension culture. Afterward, we evaluated the impact of the initial hPSC aggregate sizes (small: 86 ± 18 μm; medium: 142 ± 32 μm; large: 214 ± 34 μm) as critical bioprocess parameter on differentiation efficacy at the beginning of induction. The results indicated that 1-day priming of hPSCs as 3D aggregates (hPSpheres) with 6 μM CHIR followed by treatment with a low concentration of Activin (10 ng/mL) for 2 days resulted in efficient differentiation to definitive endoderm. This finding confirmed by the presence of ≥70% SOX17/FOXA2-double positive cells that highly expressed the anterior endodermal marker HEX. These endodermal cells differentiated efficiently into mature functional hepatocytes [60% albumin (ALB)-positive cells]. The results showed that the initial size of hPSC aggregates significantly impacted on the efficacy of differentiation. The medium sized-hPSpheres resulted in higher productivity and differentiation efficiency for scalable hepatocytes production, whereas small aggregates resulted in significant cell-loss after CHIR treatment and large aggregates had less efficacious endodermal differentiation. Differentiated cells exhibited multiple characteristics of primary hepatocytes as evidenced by expressions of liver-specific markers, indocyanine green and low-density lipoprotein uptake, and glycogen storage. Thus, this platform could be employed for scalable production of hPSC-derived hepatocytes for clinical and drug discovery applications.

The definitive endoderm (DE) is the embryonic germ layer that forms the gut tube and associated organs, including thymus, lungs, liver and pancreas. To understand how individual DE cells furnish gut organs, genetic fate mapping was performed using the Rosa26(lacZ) Cre-reporter paired with a tamoxifen-inducible DE-specific Cre-expressing transgene. We established a low tamoxifen dose that infrequently induced heritable lacZ expression in a single cell of individual E8.5 mouse embryos and identified clonal cell descendants at E16.5. As expected, only a fraction of the E16.5 embryos contained lacZ-positive clonal descendants and a subset of these contained descendants in multiple organs, revealing novel ontogeny. Furthermore, immunohistochemical analysis was used to identify lacZ-positive hepatocytes and biliary epithelial cells, which are the cholangiocyte precursors, in each clonally populated liver. Together, these data not only uncover novel and suspected lineage relationships between DE-derived organs, but also illustrate the bipotential nature of individual hepatoblasts by demonstrating that single hepatoblasts contribute to both the hepatocyte and the cholangiocyte lineage in vivo.

Differentiation of pluripotent cells into endoderm-related cell types initially requires in vitro gastrulation into the definitive endoderm (DE). Most differentiation protocols are initiated from colonies of pluripotent cells complicating their adaption due to insufficiently defined starting conditions. The protocol described here was initiated from a defined cell number of dispersed single cells and tested on three different human embryonic stem cell lines and one human induced pluripotent stem cell line. Combined activation of ActivinA/Nodal signaling and GSK3 inhibition for the first 24 h, followed by ActivinA/Nodal signaling efficiently induced the DE state. Activation of ActivinA/Nodal signaling alone was not effective. Efficient GSK3 inhibition allowed the reduction of the ActivinA concentration during the entire protocol. A feeder-independent cultivation of pluripotent cells was preferred to achieve the high efficiency and robustness since feeder cells hindered the differentiation process. Additionally, inhibition of the phosphatidylinositol 3-kinase (PI3K) signaling pathway was not required, nonetheless yielding high cell numbers efficiently committed toward the DE. Finally, the endoderm generated could be differentiated further into PDX1-positive pan-pancreatic cells and NGN3-positive endocrine progenitors. Thus, this efficient and robust DE differentiation protocol is a step forward toward better reproducibility due to the well-defined conditions based on dispersed single cells from feeder-free-cultivated human pluripotent cells.

Summary Early endoderm progenitors naturally possess robust propagating potential to develop a majority of meter-long gastrointestinal tracts and are therefore considered as a promising source for therapy. Here, we demonstrated the reproducible generation of human CDX2+ posterior gut endoderm cells (PGECs) from five induced pluripotent stem cell clones by manipulating FGF, TGF, and WNT signaling. Transcriptome analysis suggested that putative PGECs harbored an intermediate signature profile between definitive endoderm and organ-specific endoderm. We found that combinatorial EGF, VEGF, FGF2, Chir99021, and A83-01 treatments selectively amplify storable PGECs up to 1021 cell scale without any gene transduction or feeder use. PGECs, compared with induced pluripotent stem cells, showed stable differentiation propensity into multiple endodermal lineages without teratoma formation. Furthermore, transplantation of PGEC-derived liver bud organoids showed therapeutic potential against fulminant liver failure. Together, the robustly amplified PGECs may be a promising cellular source for endoderm-derived organoids in studying human development, modeling disease, and, ultimately, therapy., Graphical Abstract, Highlights • Successful activation of posteriorization program using human iPSC-derived endoderm • CDX2+ PGECs exhibit a robust amplification potential after passaging and freezing • PGECs with differentiation potential into hindgut and liver bud organoids • PGEC liver bud transplant is therapeutically effective for acute liver failure, In this article, Takebe, Taniguchi, and colleagues derived a unique population of CDX2+ posterior endoderm progenitors (PGECs) from human pluripotent stem cells that are highly expandable and storable in a chemically defined condition. CDX2+ endoderm progenitors can form multiple endodermal organoids. Transplantation of human liver bud organoids from robustly propagated PGECs rescued lethal liver failure of immunodeficient mice.

Summary Heterozygous de novo mutations in GATA6 are the most frequent cause of pancreatic agenesis in humans. In mice, however, a similar phenotype requires the biallelic loss of Gata6 and its paralog Gata4. To elaborate the human-specific requirements for GATA6, we chose to model GATA6 loss in vitro by combining both gene-edited and patient-derived pluripotent stem cells (hPSCs) and directed differentiation toward β-like cells. We find that GATA6 heterozygous hPSCs show a modest reduction in definitive endoderm (DE) formation, while GATA6-null hPSCs fail to enter the DE lineage. Consistent with these results, genome-wide studies show that GATA6 binds and cooperates with EOMES/SMAD2/3 to regulate the expression of cardinal endoderm genes. The early deficit in DE is accompanied by a significant reduction in PDX1+ pancreatic progenitors and C-PEPTIDE+ β-like cells. Taken together, our data position GATA6 as a gatekeeper to early human, but not murine, pancreatic ontogeny., Highlights • Heterozygous loss of human GATA6 results in pancreatic agenesis • GATA6+/− hPSCs form fewer pancreatic progenitors and β cells in vitro • GATA6−/− hPSCs entirely fail to form the definitive endoderm in vitro • GATA6 binds to and cooperates with EOMES/SMAD2/3 to regulate cardinal endodermal genes, In this article, Chia et al. model human pancreatic agenesis in vitro that results from heterozygous loss of the GATA6 gene. Using both gene-edited and patient-derived hPSCs, they demonstrate that GATA6 serves as a gatekeeper to early human, but not murine, pancreatic ontogeny. Genome-wide studies further show that GATA6 binds to and cooperates with EOMES/SMAD2/3 to regulate the expression of cardinal endoderm genes.

Definitive endoderm (DE) is the first stage of human pluripotent stem cell (hPSC) differentiation into hepatocyte-like cells. Developing human liver cell models for pharmaceutical applications is highly demanding. Due to the vast number of existing protocols to generate DE cells from hPSCs, we aimed to compare the specificity and efficiency of selected published differentiation conditions. We differentiated two hPSC lines (induced PSC and embryonic stem cell) to DE cells on Matrigel matrix using growth factors (Activin A and Wnt-3a) and small molecules (sodium butyrate and IDE 1) in different combinations. By studying dynamic changes during 6 days in cell morphology and the expression of markers for pluripotency, DE, and other germ layer lineages, we found that Activin A is essential for DE differentiation, while Wnt-3a and sodium butyrate are dispensable. Although sodium butyrate exerted rapid DE differentiation kinetics, it caused massive cell death and could not generate sufficient cells for further differentiation and applications. We further discover that IDE 1 could not induce DE as reported previously. Hereby, we compared different conditions for DE induction and found an effective six day-protocol to obtain DE cells for the further differentiation and applications.

Differentiation of human pluripotent stem cells towards definitive endoderm (DE) is the critical first step for generating cells comprising organs such as the gut, liver, pancreas and lung. This in-vitro differentiation process generates a heterogeneous population with a proportion of cells failing to differentiate properly and maintaining expression of pluripotency factors such as Oct4. RNA sequencing of single cells collected at four time points during a 4-day DE differentiation identified high expression of metallothionein genes in the residual Oct4-positive cells that failed to differentiate to DE. Using X-ray fluorescence microscopy and multi-isotope mass spectrometry, we discovered that high intracellular zinc level corresponds with persistent Oct4 expression and failure to differentiate. This study improves our understanding of the cellular heterogeneity during in-vitro directed differentiation and provides a valuable resource to improve DE differentiation efficiency. Keywords: hPSC, Differentiation, Definitive endoderm, Heterogeneity, Single cell, RNA sequencing

Summary Induced pluripotent stem cells were created from a pancreas agenesis patient with a mutation in GATA6. Using genome-editing technology, additional stem cell lines with mutations in both GATA6 alleles were generated and demonstrated a severe block in definitive endoderm induction, which could be rescued by re-expression of several different GATA family members. Using the endodermal progenitor stem cell culture system to bypass the developmental block at the endoderm stage, cell lines with mutations in one or both GATA6 alleles could be differentiated into β-like cells but with reduced efficiency. Use of suboptimal doses of retinoic acid during pancreas specification revealed a more severe phenotype, more closely mimicking the patient’s disease. GATA6 mutant β-like cells fail to secrete insulin upon glucose stimulation and demonstrate defective insulin processing. These data show that GATA6 plays a critical role in endoderm and pancreas specification and β-like cell functionality in humans., Highlights • GATA6 is required for definitive endoderm specification in human ES/iPS cells • Bypassing the endoderm defect allows GATA6 mutants to generate β-like cells • Suboptimal retinoic acid signaling blocks pancreas specification in GATA6 mutants • GATA6 is critical for human β cell function in vitro, In this study, Gadue and colleagues studied GATA6 mutant pluripotent stem cells and demonstrate that GATA6 is necessary for human definitive endoderm specification. GATA6 also plays an important role in pancreas specification which could be partially bypassed by retinoic acid signaling. Furthermore, derivative β cells lacked glucose responsiveness in vitro, showing that GATA6 is vital for appropriate human pancreas development.

Signalling downstream of Activin/Nodal (ActA) and Wnt can induce endoderm differentiation and also support self-renewal in pluripotent cells. Here we find that these apparently contradictory activities are fine-tuned by insulin. In the absence of insulin, the combination of these cytokines supports endoderm in a context-dependent manner. When applied to naive pluripotent cells that resemble peri-implantation embryos, ActA and Wnt induce extra-embryonic primitive endoderm (PrE), whereas when applied to primed pluripotent epiblast stem cells (EpiSC), these cytokines induce gastrulation-stage embryonic definitive endoderm. In naive embryonic stem cell culture, we find that insulin complements LIF signalling to support self-renewal; however, when it is removed, LIF, ActA and Wnt signalling not only induce PrE differentiation, but also support its expansion. Self-renewal of these PrE cultures is robust and, on the basis of gene expression, these cells resemble early blastocyst-stage PrE, a naive endoderm state able to make both visceral and parietal endoderm.

Background The definitive endoderm arises as a naive epithelial sheet that produces the entire gut tube and associated organs including the liver, pancreas and lungs. Murine explant studies demonstrate that Fibroblast Growth Factor (FGF) signaling from adjacent tissues is required to induce hepatic gene expression from isolated foregut endoderm. The requirement of FGF signaling during liver development is examined via small molecule inhibition during whole embryo culture.

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.

Human induced pluripotent stem cells (hiPSCs) are attractive sources of cells for disease modeling in vitro, and they may eventually provide access to cells/tissues for the treatment of many degenerative diseases. Stepwise differentiation from hiPSCs to definitive endoderm (DE) will identify a key step in hepatocytes and beta cell development and may prove useful for transplantation therapy for liver diseases and diabetes. Inducer of definitive endoderm 1 (IDE1) is known to play an important role in the regional specification of DE. Here, we have investigated the effect of stimulation with IDE1 on the development of hiPSCs into DE cells in three-dimensional (3D) cultures. The differentiation was determined by immunofluorescence staining with Sox17, FoxA2, and goosecoid (Gsc) and also by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis. In this study, we showed that hiPSCs with 6-day IDE1 treatment (as chemical tool) on poly(ε-caprolactone) (PCL) nanofibrous scaffold were able to differentiate into DE cells.

Activin A is a critical regulator in human embryonic stem cells (hESCs) maintenance and differentiation. Different concentrations of Activin A affect hESC maintenance and differentiation in different ways. A high concentration favors anterior primitive streak and gives rise to DE if the stimulation persists. hESCs were cultured with and without 10 ng/mL Activin A supplement respectively. The two groups of cells were differentiated into endoderm cells with 100 ng/mL Activin A and other reagents. Microarray-based DNA methylation was analyzed with the Infinium Human Methylation450 BeadChip on these two groups. There was a significant difference in endoderm differentiation efficiency (average efficiency: 71 vs. 58.5%, P

SummaryHuman pluripotent stem cell (hPSC) differentiation typically yields heterogeneous populations. Knowledge of signals controlling embryonic lineage bifurcations could efficiently yield desired cell types through exclusion of alternate fates. Therefore, we revisited signals driving induction and anterior-posterior patterning of definitive endoderm to generate a coherent roadmap for endoderm differentiation. With striking temporal dynamics, BMP and Wnt initially specified anterior primitive streak (progenitor to endoderm), yet, 24 hr later, suppressed endoderm and induced mesoderm. At lineage bifurcations, cross-repressive signals separated mutually exclusive fates; TGF-β and BMP/MAPK respectively induced pancreas versus liver from endoderm by suppressing the alternate lineage. We systematically blockaded alternate fates throughout multiple consecutive bifurcations, thereby efficiently differentiating multiple hPSC lines exclusively into endoderm and its derivatives. Comprehensive transcriptional and chromatin mapping of highly pure endodermal populations revealed that endodermal enhancers existed in a surprising diversity of “pre-enhancer” states before activation, reflecting the establishment of a permissive chromatin landscape as a prelude to differentiation.

Human-induced pluripotent stem cells (hiPSCs) are considered to be potentially able to differentiate into all human cell lineages and thus hold promise as an unlimited source for cell replacement therapies in clinical applications. Definitive endoderm (DE) formation is the first and crucial step in the development of visceral organs such as liver, lung, pancreas and so forth. Therefore, efficient generation of DE cells ensures the efficient generation of eventual target cells used in cell therapy. In the present study, Matrigel-coated poly(lactic acid)/gelatin (PLA/gelatin) nanofibrous scaffolds were utilized to investigate the proliferation and differentiation of hiPSCs into DE cells. Analyses of DE-specific markers including Sox17, FoxA2, and Gooscoid (Gsc) genes revealed higher levels of mRNA and protein expression in the differentiated hiPSCs cells cultured on PLA/gelatin scaffolds than cells differentiated in two-dimensional (2D) culture. Our results showed that three-dimensional (3D) cultures could significantly promote DE differentiation in comparison with 2D culture. Also using small molecules such as inducer of definitive endoderm 1 (IDE1) and signaling molecules such as Activin A and Wnt3a could enhance the DE differentiation of hiPSCs with Activin A/Wnt3a being significantly more potent in both 2D and 3D cultures compared to IDE1. The results of this study may have impact in tissue engineering and cell replacement therapy of visceral organs-related diseases.

Definitive endoderm (DE) differentiation from mouse embryonic stem cell (mESC) monolayer cultures has been limited by poor cell survival or low efficiency. Recently, a combination of TGFβ and Wnt activation with BMP inhibition improved DE induction in embryoid bodies cultured in suspension. Based on these observations we developed a protocol to efficiently induce DE cells in monolayer cultures of mESCs. We obtained a good cell yield with 54.92% DE induction as shown by Foxa2, Sox17, Cxcr4 and E-Cadherin expression. These DE-cells could be further differentiated into posterior foregut and pancreatic phenotypes using a culture protocol initially developed for human embryonic stem cell (hESC) differentiation. In addition, this mESC-derived DE gave rise to hepatocyte-like cells after exposure to BMP and FGF ligands. Our data therefore indicate a substantial improvement of monolayer DE induction from mESCs and support the concept that differentiation conditions for mESC-derived DE are similar to those for hESCs. As mESCs are easier to maintain and manipulate in culture compared to hESCs, and considering the shorter duration of embryonic development in the mouse, this method of efficient DE induction on monolayer will promote the development of new differentiation protocols to obtain DE-derivatives, like pancreatic beta-cells, for future use in cell replacement therapies.

Unlike Xenopus laevis, Eleutherodactylus coqui develops without a tadpole. The yolk-rich vegetal region of the embryo forms a transient nutritive tissue, the nutritional endoderm (NE). The definitive endoderm (DE) in E. coqui comes from cells closer to the animal pole in contrast to its vegetal origin in X. laevis. RNA important for initiating the endoderm specification network is absent in presumptive NE cells, raising the question whether signaling occurs in them. We explored the nature of NE and asked how differences between NE and DE cells arise. We identified differences between NE and DE that first become evident at gastrula, when NE cells become multinucleated. Nuclear β-catenin, an essential cofactor of sox 17, important for endoderm formation in X. laevis, is present in NE and DE at gastrula but remains in NE long after it is not seen in DE. We cloned E. coqui homologs of TGFβs activin b and derriere and provide evidence for their maternal expression. We also detected activin b and derriere RNAs in NE at gastrula and show that NE possesses some mesoderm-inducing activity, but it is delayed with respect to DE. Our findings indicate that altered development of NE begins at gastrula. RNAs important for mesendoderm induction and some mesoderm-inducing activity are present in NE.

Several signalling cascades are implicated in the formation and patterning of the three principal germ layers, but their precise temporal-spatial mode of action in progenitor populations remains undefined. We have used conditional gene deletion of mouse β-catenin in Sox17-positive embryonic and extra-embryonic endoderm as well as vascular endothelial progenitors to address the function of canonical Wnt signalling in cell lineage formation and patterning. Conditional mutants fail to form anterior brain structures and exhibit posterior body axis truncations, whereas initial blood vessel formation appears normal. Tetraploid rescue experiments reveal that lack of β-catenin in the anterior visceral endoderm results in defects in head organizer formation. Sox17 lineage tracing in the definitive endoderm (DE) shows a cell-autonomous requirement for β-catenin in midgut and hindgut formation. Surprisingly, wild-type posterior visceral endoderm (PVE) in midgut- and hindgut-deficient tetraploid chimera rescues the posterior body axis truncation, indicating that the PVE is important for tail organizer formation. Upon loss of β-catenin in the visceral endoderm and DE lineages, but not in the vascular endothelial lineage, Sox17 expression is not maintained, suggesting downstream regulation by canonical Wnt signalling. Strikingly, Tcf4/β-catenin transactivation complexes accumulated on Sox17 cis-regulatory elements specifically upon endoderm induction in an embryonic stem cell differentiation system. Together, these results indicate that the Wnt/β-catenin signalling pathway regulates Sox17 expression for visceral endoderm pattering and DE formation and provide the first functional evidence that the PVE is necessary for gastrula organizer gene induction and posterior axis development.

Generation of functional cells from human pluripotent stem cells (PSCs) through in vitro differentiation is a promising approach for drug screening and cell therapy. However, the observed large and unavoidable variation in the differentiation potential of different human embryonic stem cell (hESC)/induced PSC (iPSC) lines makes the selection of an appropriate cell line for the differentiation of a particular cell lineage difficult. Here, we report identification of WNT3 as a biomarker capable of predicting definitive endoderm (DE) differentiation potential of hESCs. We show that the mRNA level of WNT3 in hESCs correlates with their DE differentiation efficiency. In addition, manipulations of hESCs through WNT3 knockdown or overexpression can respectively inhibit or promote DE differentiation in a WNT3 level-dependent manner. Finally, analysis of several hESC lines based on their WNT3 expression levels allowed accurate prediction of their DE differentiation potential. Collectively, our study supports the notion that WNT3 can serve as a biomarker for predicting DE differentiation potential of hESCs., Highlights ► WNT3 level in hESCs correlates with DE differentiation efficiency ► WNT3 level in hESCs does not affect pluripotent gene expression ► Manipulation of WNT3 level in hESCs can change their DE differentiation potential.

Pancreatic progenitor (PP) cells are tissue-committed cells, which can differentiate into all kinds of pancreatic cells. They are potential candidates for regeneration of pancreatic tissue. However, it is unfeasible to acquire PP cells from pancreatic tissues and expand them in vitro. Generation of PP cells from adipose tissue-derived mesenchymal stem cells (AD-MSCs) would provide an unlimited source of PP cells. Here we developed a 2-step stepwise protocol, which induced AD-MSCs to generate FOXA2- or SOX17-positive definitive endoderm (DE) (5 days) and pancreatic and duodenal homeobox gene 1 (PDX1)-positive PP cells (4-6 days). By mimicking the developmental progress in embryonic development, we optimized the timing and combination of cytokines to activate the key signaling pathways during pancreatic development. We found that activating the Nodal/Activin signal with Activin A could induce differentiation of AD-MSCs toward DE, which could be further promoted by the Wnt signaling pathway activator Wnt3a. Besides, transient T (BRACHYURY)(+) mesendodermal cells were observed during formation of DE from AD-MSCs. Subsequently, the Wnt signaling pathway inhibitor Dkk1 along with retinoic acid/FGF2 (60 ng/mL) further induced AD-MSC-derived DE cells to differentiate into PDX1-positive PP cells. The derived PP cells were capable to form pancreatic endocrine or exocrine cells. In conclusion, we established a stepwise protocol that could derive DE and PP cells from AD-MSCs. It might provide an unlimited source of autologous PP cells for pancreatic diseases.

Despite the enormous progress in studying definitive endoderm (DE) differentiation from human embryonic stem cells (hESCs), none of the reported protocols have produced a universal, cost-effective, and competent DE with the capability to further differentiate into endodermal derivatives. In this study, by using a 2-step differentiation strategy, we have treated hESCs for 1 day with "priming" small molecules (SM), [stauprimide, NSC-308848, rapamycin (Rapa), and/or CHIR] and for the next 3 days with "inducing" SM (LY294002, cymarin, IDE1, and/or IDE2) in conjunction with activin A. In the positive control group, we treated hESCs with Wnt3a (25 ng/mL) for 1 day and activin A (100 ng/mL; W/A100-A100) for the next 3 days. Gene expression analysis showed that treatment of hESCs with 100 nM Rapa and 50 ng/mL activin A (Rapa-A50) out of 25 combinations of factors gave rise to higher expressions of 2 DE-specific genes, SOX17 and FOXA2. Similar results were obtained after treating 2 other hESC lines with this regimen. To investigate the competency of Rapa-A50-induced DE for further differentiation into endodermal derivatives, these cells and W/A100-A100-induced DE cells (positive control) were further differentiated into pancreatic progenitors (PP), then into pancreatic endocrine (PE) cells using 5 previously described differentiation protocols. Gene analysis of differentiated cells showed that the established protocols were insufficient to enable universal differentiation into PE, whereas Rapa-A50-induced DE cells were more competent for PP differentiation in a protocol-dependent manner. Additionally, Rapa-A50-induced DE had the capability to differentiate into hepatocyte-like cells (HLCs) as efficiently as W/A100-A100-induced DE. These data have indicated that hESCs primed with Rapa, and induced by a lower concentration of activin A, could lead to DE that had the capability to further differentiate into HLCs and PP cells, but not PE cells. Thus, current protocols for the differentiation of DE into PE still need additional study.

Human embryonic stem cells (hESCs) can be differentiated into multiple cell types with great therapeutic potential. However, optimizing the often multi-week cultures to obtain sufficient differentiated cell yields has been in part limited by the high variability of even parallel hESC differentiation cultures. We describe the isolation and features of a subline of CA1 hESCs (CA1S) that display a very high 25% cloning efficiency while retaining many properties of the parental hESCs, including being karyotypically normal and their ability to generate teratomas containing all three germ layers. Although more detailed analysis revealed that CA1S cells have a 3.8 Mb genomic duplication on chromosome 20, they remain highly useful. In particular, CA1S cells are readily expanded at high yields in culture and possess greatly reduced well-to-well variation even when seeded at 100 cells/well. Thus, 10(8) CA1S cells can be generated within one week from 10(6) cells to seed 10(6) wells. We determined that CA1S cells have the capacity to follow established in vitro differentiation protocols to pancreatic progenitors and subsequent hormone-positive cell types and used CA1S cells to explore definitive endoderm induction in a high performance screen (Z-factor = 0.97). This system revealed that CA1S cells do not require WNT3A to efficiently form definitive endoderm, a finding that was confirmed with H1 hESCs, although H1 cells did show modest benefits of high WNT3A doses. Proliferative index measurements of CA1S cells were shown to rapidly reflect their differentiation status in a high throughput system. Though results obtained with CA1S cells will need to be confirmed using conventional hESC lines, these cells should ease the development of optimized hESC growth and differentiation protocols. In particular, they should limit the more arduous secondary screens using hESCs to a smaller number of variables and doses.

Pluripotent stem cells have the capability to differentiate into any somatic cell type of the human body. The generation of surrogate cells for the treatment of liver, lung, and pancreatic diseases is of great medical interest. First, the in vitro formation into cells of the definitive endoderm is required. Upon commitment into this lineage, the cells express transcription factors such as FOXA2, SOX17, HNF1B; GATA family members; and the surface protein CXCR4. Unfortunately, some pluripotent stem cells resist the differentiation and contaminate the culture. Thus, we describe here an endoderm differentiation protocol, which yields endoderm-committed cells in high numbers in a 4-day treatment protocol. Second, a method for the purification of CXCR4-positive endoderm cells by magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS) is described. The purification by MACS is quick and reliable and can be used to obtain pure endoderm cells either meant for downstream analysis such as omics or further differentiation experiments into endoderm-derived somatic cells. © 2017 by John Wiley & Sons, Inc. Keywords: human pluripotent stem cells; differentiation; definitive endoderm; MACS; FACS; purification; cell sorting

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.

Protocols 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., Summary: Using the differentiation of huESCs as a model for endoderm formation, we reveal that the transcription factor GATA6 regulates the onset of endoderm gene expression and is required for its viability.