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Genetic analysis of an adult patient with an unusual course of Ketosis-Prone Diabetes (KPD) and lacking islet autoantibodies demonstrated a nucleotide variant in the 5’-UTR of PDX1, a beta-cell development gene. When differentiated to the pancreatic lineage, his induced pluripotent stem cells stalled at the definitive endoderm stage. Metabolomic analysis of the cells revealed that this was associated with leucine hypersensitivity during transition from the definitive endoderm to the pancreatic progenitor stage, and RNA-sequencing showed defects in leucine-sensitive mTOR pathways contribute to the differentiation deficiency. CRISPR-Cas9 manipulation of the PDX1 variant demonstrated that it is necessary and sufficient to confer leucine sensitivity and the differentiation block, likely due to disruption of binding of the transcriptional regulator NFY to the PDX1 5’-UTR, leading to decreased PDX1 expression at the early pancreatic progenitor stage. Thus, the combination of an underlying defect in leucine catabolism characteristic of KPD with a functionally relevant heterozygous variant in a critical beta-cell gene that confers increased leucine sensitivity and inhibits endocrine cell differentiation resulted in the phenotype of late-onset beta-cell failure in this patient. We define the molecular pathogenesis of a diabetes syndrome and demonstrate the power of multi-omics analysis of patient-specific stem cells for clinical discovery.

Background Cell-specific and developmental mechanisms contribute to expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, however, its developmental regulation is poorly understood. Here we use human induced pluripotent stem cells (hiPSCs) differentiated into pseudostratified airway epithelial cells to study these mechanisms. Results Changes in gene expression and open chromatin profiles were investigated by RNA-seq and ATAC-seq, and revealed that alterations in CFTR expression are associated with differences in stage-specific open chromatin. Additionally, 2 novel open chromatin regions, at +19.6kb and +22.6kb 3' to the CFTR translational stop signal, were observed in definitive endoderm (DE) cells, prior to an increase in CFTR expression in anterior foregut endoderm (AFE) cells. Chromatin studies in DE and AFE cells revealed enrichment of active enhancer marks and occupancy of OTX2 at these sites in DE cells. Loss of OTX2 in DE cells alters histone modifications across the CFTR locus and results in a 2.5-fold to 5-fold increase in CFTR expression. However, deletion of the +22.6kb site alone does not affect CFTR expression in DE or AFE cells. Conclusions These results suggest that a network of interacting cis-regulatory elements recruit OTX2 to the locus to impact CFTR expression during early endoderm differentiation. This article is protected by copyright. All rights reserved.

Scalable processes are requisite for the robust biomanufacturing of human pluripotent stem cell (hPSC)-derived therapeutics. Toward this end, we demonstrate the xeno-free expansion and directed differentiation of human embryonic (hESCs) and induced pluripotent cells (hiPSCs) to definitive endoderm (DE) in a controlled stirred suspension bioreactor (SSB). Based on previous work on converting hPSCs to insulin-producing progeny, differentiation of two hPSC lines was optimized in planar cultures yielding up to 87% FOXA2+ /SOX17+ cells. Next, hPSCs were propagated in a SSB with controlled pH and dissolved oxygen. Cultures displayed a 10- to 12-fold increase in cell number over 5-6 days with maintenance of pluripotency (>85% OCT4+ ) and viability (>85%). For differentiation, SSB cultures yielded up to 89% FOXA2+ /SOX17+ cells or ~ 8 DE cells per seeded hPSC. Specification to DE cell fate was consistently more efficient in the bioreactor compared to planar cultures. Hence, a tunable strategy is established that is suitable for the xeno-free manufacturing of DE cells from different hPSC lines in scalable SSBs. This work advances bioprocess development for producing a wide gamut of human DE cell-derived therapeutics. This article is protected by copyright. All rights reserved.

Summary The ability of human induced pluripotent stem cells (hiPSCs) to differentiate in vitro to each of the three germ layer lineages has made them an important model of early human development and a tool for tissue engineering. However, the factors that disturb the intricate transcriptional choreography of differentiation remain incompletely understood. Here, we uncover a critical time window during which DNA damage significantly reduces the efficiency and fidelity with which hiPSCs differentiate to definitive endoderm. DNA damage prevents the normal reduction of p53 levels as cells pass through the epithelial-to-mesenchymal transition, diverting the transcriptional program toward mesoderm without induction of an apoptotic response. In contrast, TP53-deficient cells differentiate to endoderm with high efficiency after DNA damage, suggesting that p53 enforces a “differentiation checkpoint” in early endoderm differentiation that alters cell fate in response to DNA damage., Highlights • DNA damage impairs the efficiency and fidelity of human stem cell differentiation • p53 is reduced transiently during the commitment to definitive endoderm • Preventing p53 reduction diverts cells away from endoderm without apoptosis • p53-deficient cells differentiate to endoderm efficiently in the face of DNA damage, Eldridge et al. demonstrate a temporal window during which the differentiation of human iPSCs to definitive endoderm is sensitive to low levels of DNA damage, causing diversion of the differentiation program toward a more mesodermal fate. This effect is almost entirely dependent on p53, with both the efficiency and the fidelity of endoderm differentiation being restored in damaged p53-deficient cells.

SummaryImproving methods for human embryonic stem cell differentiation represents a challenge in modern regenerative medicine research. Using drug repurposing approaches, we discover small molecules that regulate the formation of definitive endoderm. Among them are inhibitors of known processes involved in endoderm differentiation (mTOR, PI3K, and JNK pathways) and a new compound, with an unknown mechanism of action, capable of inducing endoderm formation in the absence of growth factors in the media. Optimization of the classical protocol by including this compound achieves the same differentiation efficiency with a 90% cost reduction. The gene expression profile induced by the compound suggests that it is an inhibitor of the MYC pathway. The proposed in silico procedure for candidate molecule selection has broad potential for improving stem cell differentiation protocols.

Mesendoderm cells are key intermediate progenitors that form at the early primitive streak (PrS) and give rise to mesoderm and endoderm in the gastrulating embryo. We have identified an interaction between CNOT3 and the cell cycle kinase Aurora B that requires sequences in the NOT box domain of CNOT3 and regulates MAPK/ERK signaling during mesendoderm differentiation. Aurora B phosphorylates CNOT3 at two sites located close to a nuclear localization signal and promotes localization of CNOT3 to the nuclei of mouse embryonic stem cells (ESCs) and metastatic lung cancer cells. ESCs that have both sites mutated give rise to embryoid bodies that are largely devoid of mesoderm and endoderm and are composed mainly of cells with ectodermal characteristics. The mutant ESCs are also compromised in their ability to differentiate into mesendoderm in response to FGF2, BMP4, and Wnt3 due to reduced survival and proliferation of differentiating mesendoderm cells. We also show that the double mutation alters the balance of interaction of CNOT3 with Aurora B and with ERK and reduces phosphorylation of ERK in response to FGF2. Our results identify a potential adaptor function for CNOT3 that regulates the Ras/MEK/ERK pathway during embryogenesis.

Specification of the primary heart field in mouse embryos requires signaling from the anterior visceral endoderm (AVE). The nature of these signals is not known. We hypothesized that the TGFβ-activated kinase (TAK1/Map3k7) may act as a cardiogenic factor, based on its expression in heart-inducing endoderm and its requirement for cardiac differentiation of p19 cells. To test this, mouse embryonic stem (ES) cells overexpressing Map3k7 were isolated and differentiated as embryoid bodies (EBs). Map3k7-overexpressing EBs showed increased expression of AVE markers but interestingly, showed little effect on mesoderm formation and had no impact on overall cardiomyocyte formation. To test whether the pronounced expansion of endoderm masks an expansion of cardiac lineages, chimeric EBs were made consisting of Map3k7-overexpressing ES and wild type ES cells harboring a cardiac reporter transgene, MHCα::GFP, allowing cardiac differentiation to be assessed specifically in wild type ES cells. Wild type ES cells co-cultured with Map3k7-overexpressing cells had a 4-fold increase in expression of the cardiac reporter, supporting the hypothesis that Map3k7 increases the formation of cardiogenic endoderm. To further examine the role of Map3k7 in early lineage specification, other endodermal markers were examined. Interestingly, markers that are expressed in both the VE and later in gut development were expanded, whereas transcripts that specifically mark the early definitive (streak-derived) endoderm (DE) were not. To determine if Map3k7 is necessary for endoderm differentiation, EBs were grown in the presence of the Map3k7 specific inhibitor 5Z-7-oxozeaenol. Endoderm differentiation was dramatically decreased in these cells. Western blot analysis showed that known downstream targets of Map3k7 (Jnk, Nemo-like kinase (NLK) and p38 MAPK) were all inhibited. By contrast, transcripts for another TGFβ target, Sonic Hedgehog (Shh) were markedly upregulated, as were transcripts for Gli2 (but not Gli1 and Gli3). Together these data support the hypothesis that Map3k7 governs the formation, or proliferation of cardiogenic endoderm.

Summary Human pluripotent stem cells (hPSCs) are an essential cell source in tissue engineering, studies of development, and disease modeling. Efficient, broadly amenable protocols for rapid lineage induction of hPSCs are of great interest in the stem cell biology field. We describe a simple, robust method for differentiation of hPSCs into mesendoderm in defined conditions utilizing single-cell seeding (SCS) and BMP4 and Activin A (BA) treatment. BA treatment was readily incorporated into existing protocols for chondrogenic and endothelial progenitor cell differentiation, while fine-tuning of BA conditions facilitated definitive endoderm commitment. After prolonged differentiation in vitro or in vivo, BA pretreatment resulted in higher mesoderm and endoderm levels at the expense of ectoderm formation. These data demonstrate that SCS with BA treatment is a powerful method for induction of mesendoderm that can be adapted for use in mesoderm and endoderm differentiation., Highlights • Single-cell seeding with BMP4/Activin A treatment supports hPSC mesendoderm induction • The mesendoderm protocol is amenable to mesoderm and endoderm lineage differentiation • Mesoderm/endoderm formation was enhanced in basal conditions in vitro and in vivo, In this article, Carpenedo and Stanford and colleagues demonstrate a robust and reproducible single-cell seeding method for rapid induction of mesendoderm for hPSCs. Transcriptomic data indicated that the method could be applied to mesoderm and endoderm differentiation protocols, which was demonstrated experimentally. Formation of mesoderm and endoderm following pre-differentiation was further demonstrated in long-term in vitro and in vivo assays.

Publisher Copyright: © Copyright © 2021 Bogacheva, Harjumäki, Flander, Taalas, Bystriakova, Yliperttula, Xiang, Leung and Lou. The generation of human stem cell-derived spheroids and organoids represents a major step in solving numerous medical, pharmacological, and biological challenges. Due to the advantages of three-dimensional (3D) cell culture systems and the diverse applications of human pluripotent stem cell (iPSC)-derived definitive endoderm (DE), we studied the influence of spheroid size and 3D cell culture systems on spheroid morphology and the effectiveness of DE differentiation as assessed by quantitative PCR (qPCR), flow cytometry, immunofluorescence, and computational modeling. Among the tested hydrogel-based 3D systems, we found that basement membrane extract (BME) hydrogel could not retain spheroid morphology due to dominant cell–matrix interactions. On the other hand, we found that nanofibrillar cellulose (NFC) hydrogel could maintain spheroid morphology but impeded growth factor diffusion, thereby negatively affecting cell differentiation. In contrast, suspension culture provided sufficient mass transfer and was demonstrated by protein expression assays, morphological analyses, and mathematical modeling to be superior to the hydrogel-based systems. In addition, we found that spheroid size was reversely correlated with the effectiveness of DE formation. However, spheroids of insufficient sizes failed to retain 3D morphology during differentiation in all the studied culture conditions. We hereby demonstrate how the properties of a chosen biomaterial influence the differentiation process and the importance of spheroid size control for successful human iPSC differentiation. Our study provides critical parametric information for the generation of human DE-derived, tissue-specific organoids in future studies.

The potential functions of hepatocyte-like cells (HLCs) derived from human embryonic stem cells (hESCs) hold great promise for disease modeling and drug screening applications. Provided here is an efficient and reproducible method for differentiation of hESCs into functional HLCs. The establishment of an endoderm lineage is a key step in the differentiation to HLCs. By our method, we regulate the key signaling pathways by continuously supplementing Activin A and CHIR99021 during hESC differentiation into definitive endoderm (DE), followed by generation of hepatic progenitor cells, and finally HLCs with typical hepatocyte morphology in a stagewise method with completely defined reagents. The hESC-derived HLCs produced by this method express stage-specific markers (including albumin, HNF4α nuclear receptor, and sodium taurocholate cotransporting polypeptide (NTCP)), and show special characteristics related to mature and functional hepatocytes (including indocyanine green staining, glycogen storage, hematoxylin-eosin staining and CYP3 activity), and can provide a platform for the development of HLC-based applications in the study of liver diseases.

Summary Human pluripotent stem cell (hPSC)-derived pancreatic progenitors (PPs) provide promising cell therapies for type 1 diabetes. Current PP differentiation requires a high amount of Activin A during the definitive endoderm (DE) stage, making it economically difficult for commercial ventures. Here we identify a dose-dependent role for Wnt signaling in controlling DE differentiation without Activin A. While high-level Wnt activation induces mesodermal formation, low-level Wnt activation by a small-molecule inhibitor of glycogen synthase kinase 3 is sufficient for DE differentiation, yielding SOX17+FOXA2+ DE cells. BMP inhibition further enhances this DE differentiation, generating over 87% DE cells. These DE cells could be further differentiated into PPs and functional β cells. RNA-sequencing analysis of PP differentiation from hPSCs revealed expected transcriptome dynamics and new gene regulators during our small-molecule PP differentiation protocol. Overall, we established a robust growth-factor-free protocol for generating DE and PP cells, facilitating scalable production of pancreatic cells for regenerative applications., Graphical abstract, Highlights • Differential activation of the Wnt pathway in hPSCs leads to mesoderm or endoderm fate • BMP signaling inhibition enhances CHIR99021-induced DE differentiation from hPSCs • Small-molecule DE cells can further become pancreatic progenitors and β cells • RNA-seq of small-molecule differentiation revealed expected transcriptome changes, In this article, Lian and colleagues identify a dose-dependent role for Wnt signaling in differentiating hPSCs. While high-level Wnt activation induces mesoderm, low-level Wnt activation is sufficient to yield definitive endoderm (DE) cells. BMP signaling inhibition further enhances this differentiation. The authors further establish a small-molecule protocol to differentiate DE cells into pancreatic progenitors, which could be further differentiated into functional β cells.

Understanding the development of human respiratory tissues is crucial for modeling and treating lung disorders. The molecular details for the specification of lung progenitors from human pluripotent stem cells (hPSCs) are unclear. Here, we use single cell RNA-sequencing with high temporal resolution along an optimized differentiation protocol to determine the transcriptional hierarchy of lung specification from human hPSCs and map out the underlying single cell trajectories. We show that Sonic hedgehog, TGF-βand Notch activation are required in anISL1/NKX2-1trajectory that gives rise to lung progenitors during the foregut endoderm stage. Induction ofHHEXmarks an alternative trajectory at the early definitive endoderm stage, which precedes the lung trajectory and generates a major hepatoblast population. Moreover, neither KDR+ nor mesendoderm progenitors are apparent intermediate states of lung and hepatic lineages. Our hierarchical multistep model predicts mechanisms leading to lung organogenesis, and creates a basis for studying early human lung development, as well as hPSC based disease and drug research.Abstract Figure

LIM kinases are involved in various cellular events such as migration, cycle, and differentiation, but whether they have a role in the specification of mammalian early endoderm remains unclear. In the present study, we found that depletion of LIMK2 severely inhibited the generation of definitive endoderm (DE) from human embryonic stem cells (hESCs) and promoted an early neuroectodermal fate. Upon the silencing of LIMK2 during the endodermal differentiation, the assembly of actin stress fibers was disturbed, and the phosphorylation of cofilin was decreased. In addition, knockdown of LIMK2 during DE differentiation also interfered the upregulation of epithelial-to-mesenchymal transition (EMT)-related genes and cell migration. Collectively, the results highlight that the serine/threonine kinase LIMK2, acting as a key regulator in actin remodeling, plays a critical role in endodermal lineage determination.

Therapeutic use of differentiated organ cells from human induced pluripotent stem cells (hiPSCs) is one of the promising strategies for regenerative medicine. Differentiation into definitive endoderm is an essential process in the preparation of metabolic organs. However, the manufacturability of differentiation is limited due to the high-cost cytokines required for the differentiation of endodermal lineage. Furthermore, the cytokines remaining in the used culture medium and possible endogenous factors are removed along with toxic metabolites by the medium replacement. To address these problems, the application of dialysis culture can retain and fully utilize their accumulation to create a better culture environment that contributes to differentiation cost reduction.

The segregation of definitive endoderm (DE) from bi-potent mesendoderm progenitors leads to the formation of two distinct germ layers. Dissecting DE commitment and onset has been challenging as it occurs within a narrow spatio-temporal window in the embryo. Here we employ a novel dual Bra/Sox17 reporter cell line to study DE onset dynamics. We find Sox17 expression initiates in a few isolated cells in vivo within a temporally restricted window. Using 2D and 3D in vitro models, we show that DE cells emerge from mesendoderm progenitors at a temporally regular, but spatially stochastic pattern, which is subsequently arranged by self-sorting of Sox17+ cells. A subpopulation of Bra-high cells commits to a Sox17+ fate independent of external Wnt signal. Self-sorting coincides with up-regulation of E-cadherin but is not necessary for DE differentiation or proliferation. Our in vivo and in vitro results highlight basic rules governing DE onset and patterning through the commonalities and differences between these systems.

Activin A plays a central role in the differentiation of stem cells into definitive endoderm, the first step in embryonic development and function development in many organ systems. The aims of this study were to induce controlled and fine-tuned cell differentiation using a gradient nanotechnology and compare this with a classic protocol and to investigate how induced pluripotent stem cells differentiated depending on the gradual increase of Activin A. The density difference was tested by attaching Activin A to a gold nanoparticle gradient for high-precision density continuity. Cells expressed the definitive endoderm markers SRY-box transcription factor 17 and transcription factor GATA-4 to different extents along the gradient, indicating a density-dependent cell response to Activin A. In both the gradient and the classic differentiation setups, the protein expression increased from days 1 to 5, but a significant increase already on day 3 was found only in the gradient-based setup. By utilizing the gradient technology to present the right amount of active biomolecules to cells in vitro, we were able to find an optimal setting for differentiation into definitive endoderm. The use of gradient surfaces for differentiation allows for improvements, such as efficiency and faster differentiation, compared with a classic protocol.

The transcription factor FoxO1 has been shown to dynamically regulate cell fate across diverse cell types. Here, we employ a human induced pluripotent stem cell (hiPSC)-to-hepatocyte differentiation system that recapitulates the process of hepatocyte specification and differentiation in the human embryo to investigate FoxO1 as a participant in the molecular events required to execute the initial stages of liver development. We demonstrate that FoxO1 is expressed in hiPSC and at all stages of hepatocyte differentiation: definitive endoderm, specified hepatocytes, immature hepatoblasts, and mature hepatocyte-like cells. Disruption of FoxO1 activity by addition of the small molecule inhibitor AS1842856 at the beginning of the differentiation protocol abolishes the formation of definitive endoderm, as indicated by the loss of endoderm gene expression and the gain in expression of multiple mesoderm genes. Moreover, we show that FoxO1 binds to the promoters of two genes with important roles in endoderm differentiation whose expression is significantly downregulated in AS1842856 treated versus untreated cells. These findings reveal a new role for FoxO1 as an essential transcriptional regulator for the establishment of definitive endoderm in humans.

Alternative splicing is critical for development; however, its role in the specification of the three embryonic germ layers is poorly understood. By performing RNA-Seq on human embryonic stem cells (hESCs) and derived definitive endoderm, cardiac mesoderm, and ectoderm cell lineages, we detect distinct alternative splicing programs associated with each lineage. The most prominent splicing program differences are observed between definitive endoderm and cardiac mesoderm. Integrative multi-omics analyses link each program with lineage-enriched RNA binding protein regulators, and further suggest a widespread role for Quaking (QKI) in the specification of cardiac mesoderm. Remarkably, knockout of QKI disrupts the cardiac mesoderm-associated alternative splicing program and formation of myocytes. These changes arise in part through reduced expression of BIN1 splice variants linked to cardiac development. Mechanistically, we find that QKI represses inclusion of exon 7 in BIN1 pre-mRNA via an exonic ACUAA motif, and this is concomitant with intron removal and cleavage from chromatin. Collectively, our results uncover alternative splicing programs associated with the three germ lineages and demonstrate an important role for QKI in the formation of cardiac mesoderm.

Background: Induced pluripotent stem cells (iPSCs) have the ability to proliferate indefinitely and differentiate into three germ layers of ectoderm, mesoderm, and endoderm. Definitive induction is the first and the most delicate stage of differentiation of various iPSC-derived organs. It has been found that the Wnt signaling pathway implicates in embryogenesis, organogenesis, and cell communication. Objectives: In the present study, we aimed to investigate the expression pattern of the Wnt5a gene as an indicator of non-canonical Wnt signaling activity during definitive endoderm induction of iPSCs. Methods: Human iPSCs (RSCB0042) were acquired from Royan stem cell bank of Royan Institute (Tehran, Iran). The iPSCs were cultured on a feeder layer of mitomycin-inactivated mouse embryonic fibroblasts (MEF), and iPSC colonies were collected for embryoid body (EB) generation by suspension culture method. Then endoderm induction step was performed using a series of small molecules. The quantitative real-time PCR was used to assess the mRNA expression of wnt5a, Nanog, OCT4, SOX17, and FOXA2 genes. Results: The production of efficient EBs confirmed by a decrease in Nanog and Oct4 gene expression and the success of DE (definite endoderm) induction step was confirmed by a high expression level of DE specific genes, Sox17, and FoxA2. A significant upregulation of Wnt5a in EB samples and a minor decrease at day 4 was observed. However, the differentiation process followed by an incremental fashion in Wnt5a mRNA expression starting from day 4 of differentiation among the samples of days 6 and 8 (DE stage). Conclusions: Our results suggest that Wnt5a is more activated at the later steps of endoderm induction rather than the early steps, which may be due to the stimulation of canonical Wnt signaling. Finding the expression level of Wnt5a could rise insights for developing more efficient differentiation induction protocols.

Lineage specification is governed by gene regulatory networks (GRNs) that integrate the activity of signaling effectors and transcription factors (TFs) on enhancers. Sox17 is a key transcriptional regulator of definitive endoderm development, and yet, its genomic targets remain largely uncharacterized. Here, using genomic approaches and epistasis experiments, we define the Sox17-governed endoderm GRN inXenopusgastrulae. We show that Sox17 functionally interacts with the canonical Wnt pathway to specify and pattern the endoderm while repressing alternative mesectoderm fates. Sox17 and β-catenin co-occupy hundreds of key enhancers. In some cases, Sox17 and β-catenin synergistically activate transcription apparently independent of Tcfs, whereas on other enhancers, Sox17 represses β-catenin/Tcf-mediated transcription to spatially restrict gene expression domains. Our findings establish Sox17 as a tissue-specific modifier of Wnt responses and point to a novel paradigm where genomic specificity of Wnt/β-catenin transcription is determined through functional interactions between lineage-specific Sox TFs and β-catenin/Tcf transcriptional complexes. Given the ubiquitous nature of Sox TFs and Wnt signaling, this mechanism has important implications across a diverse range of developmental and disease contexts.

SUMMARY In addition to driving specific gene expression profiles, transcriptional regulators are becoming increasingly recognized for their capacity to modulate chromatin structure. GATA6 is essential for the formation of definitive endoderm; however, the molecular basis defining the importance of GATA6 to endoderm commitment is poorly understood. The members of the GATA family of transcription factors have the capacity to bind and alter the accessibility of chromatin. Using pluripotent stem cells as a model of human development, we reveal that GATA6 is integral to the establishment of the endoderm enhancer network via the induction of chromatin accessibility and histone modifications. We additionally identify the chromatin-modifying complexes that interact with GATA6, defining the putative mechanisms by which GATA6 modulates chromatin architecture. The identified GATA6-dependent processes further our knowledge of the molecular mechanisms that underpin cell-fate decisions during formative development., Graphical abstract, In brief Using a hiPSC model of early development, Heslop et al. report that GATA6 is essential for chromatin patterning during definitive endoderm formation. The authors additionally identify the chromatin remodeling complexes that interact with GATA6 and that endoderm-enriched transcription factor binding profiles are significantly perturbed in the absence of GATA6.

Differentiation of pluripotent stem cells into functional parathyroid-like cells would accelerate development of important therapeutic options for subjects with parathyroid-related disorders, from the design and screening of novel pharmaceutical agents to the development of durable cellular therapies. We have established a highly reproducible directed differentiation approach leading to PTH-expressing cells from human embryonic stem cells and induced pluripotent stem cells. We accomplished this through the comparison of multiple different basal media, the inclusion of the CDK inhibitor PD0332991 in both definitive endoderm and anterior foregut endoderm stages, and a 2-stage pharyngeal endoderm series. This is the first protocol to reproducibly establish PTH-expressing cells from human pluripotent stem cells and represents a first step toward the development of functional parathyroid cells with broad applicability for medicinal and scientific investigation.

Directed in vitro differentiation of pluripotent stem cells toward definitive endoderm (DE) offers great research and therapeutic potential since these cells can further differentiate into cells of the respiratory and gastrointestinal tracts, as well as associated organs such as pancreas, liver, and thyroid. We hypothesized that culturing mouse embryonic stem cells (mESCs) under simulated microgravity (SMG) conditions in rotary bioreactors (BRs) will enhance the induction of directed DE differentiation. To test our hypothesis, we cultured the cells for 6 days in two-dimensional monolayer colony cultures or as embryoid bodies (EBs) in either static conditions or, dynamically, in the rotary BRs. We used flow cytometry and quantitative polymerase chain reaction to analyze the expression of marker proteins and genes, respectively, for pluripotency (

Advances in human pluripotent stem cell (hPSC) biology now allow the generation of organoids that resemble different regions of the gastrointestinal tract. Generation of region-specific organoids has been facilitated by developmental biology studies carried out in model organisms such as mouse, frog and chick. By mimicking embryonic development, hPSC-derived human colonic organoids (HCOs) can be generated through a stepwise differentiation, first into definitive endoderm (DE), then into mid/hindgut spheroids which are then patterned into posterior gut tissue which gives rise to HCOs following prolonged in vitro culture. HCOs undergo transitions similar to those observed in the developing colon of model organisms and human embryos. HCOs develop into tissue that resembles fetal colon on the basis of morphology, gene expression and presence of differentiated cell types. Generation of HCOs without the proper training or expertise can be a daunting task. Here, we describe a detailed protocol for differentiating hPSCs into HCOs, we include suggestions for troubleshooting these differentiations, and we discuss experimental design considerations. We have also highlighted the key advantages and limitations of the system.

The endodermal germ layer gives rise to respiratory epithelium, hepatocytes, pancreatic cells and intestinal lineages, among other cell types. These lineages can be differentiated from human pluripotent stem cells (hPSCs) via a common definitive endoderm (DE) intermediate that is characterized by the co-expression of the cell surface markers CXCR4, c-KIT and EPCAM and the transcription factors SOX17 and FOXA2. Here we provide a detailed protocol for mass production of DE from hPSCs in scalable and easy-to-handle suspension culture using a rotating Erlenmeyer flask or a sophisticated, fully controllable, 150-ml stirred tank bioreactor. This protocol uses two different media formulations that are chemically defined and xeno free and therefore good manufacturing practice ready. Our protocol allows for efficient hPSC-derived DE specification in multicellular aggregates within 3 days and generates up to 1 × 108 DE cells with >92% purity in one differentiation batch when using the bioreactor. The hPSC-derived DE cells that are generated can be cryopreserved for later downstream differentiation into various endodermal lineages. This protocol should facilitate the flexible production of mature DE derivatives for physiologically relevant disease models, high-throughput drug screening, toxicology testing and cellular therapies. The authors describe a process for the mass production and cryopreservation of definitive endodermal cells derived from human pluripotent stem cells using a chemically defined, xeno-free suspension culture in stirred tank bioreactors or rotating Erlenmeyer flasks.

Efficiency of the induction protocol is crucial for the generation of insulin-producing cells (IPCs) from human dental pulp stem cells (hDPSCs). Here, we established the integrative induction protocol by merging genetic manipulation technique with our previous published 3-step induction protocol aiming to enhance the pancreatic progenitor commitment and production yield. We found that the overexpression of PDX1 following with 3-step induction protocol were able to generate the 3-dimensional (3D) colony structure of pancreatic progenitors (PPs) with the beneficial trends of pancreatic endoderm commitment and production yield, while other protocols using the prolong maintenance of PDX1-overexpressed hDPSCs and the PDX1 overexpression after definitive endoderm induction were unable to generate and sustain the 3D structure of the colonies. Further Notch signaling manipulation by DAPT treatment showed lesser degree of positive effects on progenitor commitment and production yield. Although the generated PPs from the integrative protocol expressed pancreatic mRNA markers along with pro-insulin and insulin proteins, they still contained the defective glucose-responsive C-peptide secretion. Only basal secreted C-peptide level was observed. In summary, the integrative induction protocol potentially enhanced the PP generation with high colony production yield and could serve as an efficient platform for further hDPSC-derived IPC production and maturation.

Defining the precise regionalization of specified definitive endoderm progenitors is critical for understanding the mechanisms underlying the generation and regeneration of respiratory and digestive organs, yet the patterning of endoderm progenitors remains unresolved, particularly in humans. We performed single-cell RNA sequencing on endoderm cells during the early somitogenesis stages in mice and humans. We developed molecular criteria to define four major endoderm regions (foregut, lip of anterior intestinal portal, midgut, and hindgut) and their developmental pathways. We identified the cell subpopulations in each region and their spatial distributions and characterized key molecular features along the body axes. Dorsal and ventral pancreatic progenitors appear to originate from the midgut population and follow distinct pathways to develop into an identical cell type. Finally, we described the generally conserved endoderm patterning in humans and clear differences in dorsal cell distribution between species. Our study comprehensively defines single-cell endoderm patterning and provides novel insights into the spatiotemporal process that drives establishment of early endoderm domains.

Recent developments in stem cell biology have enabled the study of cell fate decisions in early human development that are impossible to study in vivo. However, understanding how development varies across individuals and, in particular, the influence of common genetic variants during this process has not been characterised. Here, we exploit human iPS cell lines from 125 donors, a pooled experimental design, and single-cell RNA-sequencing to study population variation of endoderm differentiation. We identify molecular markers that are predictive of differentiation efficiency of individual lines, and utilise heterogeneity in the genetic background across individuals to map hundreds of expression quantitative trait loci that influence expression dynamically during differentiation and across cellular contexts., Studying the genetic effects on early stages of human development is challenging due to a scarcity of biological material. Here, the authors utilise induced pluripotent stem cells from 125 donors to track gene expression changes and expression quantitative trait loci at single cell resolution during in vitro endoderm differentiation.

Endoderm precursors expressing FoxA2 and Sox17 develop from the epiblast through the gastrulation process. In this study, we developed an experimental system to model the endoderm‐generating gastrulation process using epiblast stem cells (EpiSCs). To this end, we established an EpiSC line i22, in which enhanced green fluorescent protein is coexpressed with Foxa2. Culturing i22 EpiSCs as aggregates for a few days was sufficient to initiate Foxa2 expression, and further culturing of the aggregates in Matrigel promoted the sequential activation of transcription factor genes involved in endoderm precursor development, e.g., Eomes, Gsc, and Sox17. In aggregation culture of i22 cells for 3 days, all cells expressed POU5F1, SOX2, and E‐cadherin, a signature of the epiblast, whereas expression of GATA4 and SOX17 was also activated moderately in dispersed cells, suggesting priming of these cells to endodermal development. Embedding the aggregates in Matrigel for further 3 days elicited migration of the cells into the lumen of laminin‐rich matrices covering the aggregates, in which FOXA2 and SOX17 were expressed at a high level with the concomitant loss of E‐cadherin, indicating the migratory phase of endodermal precursors. Prolonged culturing of the aggregates generated three segregating cell populations found in post‐gastrulation stage embryos: (1) definitive endoderm co‐expressing high SOX17, GATA4, and E‐cadherin, (2) mesodermal cells expressing a low level of GATA4 and lacking E‐cadherin, and (3) primed epiblast cells expressing POU5F1, SOX2 without E‐cadherin. Thus, aggregation of EpiSCs followed by embedding of aggregates in the laminin‐rich matrix models the gastrulation‐dependent endoderm precursor development., Changes in the distribution of SOX17‐expressing cells in the EpiSC aggregates. The SOX17‐expressing cells initially arises at scattered positions in simple aggregates of EpiSCs. Embedding the aggregates in laminin‐rich Matrigel increased the fraction of SOX17‐expressing endoderm precursor cells, and these SOX17‐expressing cells migrated out in the laminin‐rich mantle zone, and eventually settled in the mantle zone forming epitheloid endodermal tissues.

Surface topography can have significant impact on the cell fate decisions of stem cells. Construction of a biomaterial that can mimic the physiological microenvironment can be used to enhance the maintenance or differentiation of stem cells. Here, we manufacture two surfaces with monolayer binary colloidal crystals (BCCs) having different topographies, Si2PM and Si5PM, and examined their effect on maintenance and differentiation of human embryonic stem cells (hESCs). Our results demonstrated that BCCs can be coupled with vitronectin to maintain pluripotency of hESCs during culture, and with a gelatin coating to improve early definitive endoderm differentiation of hESCs using an embryonic body assay (i.e., FOXA2 up-regulation). BCCs can be used as novel cell culture substrates to support stem cell maintenance and differentiation.

Tfcp2l1 is a transcription factor critical for mouse embryonic stem cell (mESC) maintenance. However, its role in human ESCs (hESCs) remains unclear. Here, we investigated the functions of Tfcp2l1 in controlling hESC activity and showed that Tfcp2l1 is functionally important in the maintenance of hESC identity. Tfcp2l1 expression is highly enriched in hESCs and dramatically decreases upon differentiation. Forced expression of Tfcp2l1 promoted hESC self-renewal. Functional analysis of the mutant forms of Tfcp2l1 revealed that both the CP2- and SAM-like domains are indispensable for Tfcp2l1 to maintain the undifferentiated state of hESCs. Notably, the CP2-like domain is closely related to the suppression of definitive endoderm and mesoderm commitment. Accordingly, knockdown of Tfcp2l1 significantly induced differentiation preferentially into definitive endoderm and mesoderm. Further studies found that inhibition of Wnt/β-catenin signaling pathway by IWR1 is able to eliminate the differentiation caused by Tfcp2l1 downregulation. Taken together, these findings reveal the unique and crucial role of Tfcp2l1 in the determination of hESC fate and will expand our understanding of the self-renewal and differentiation circuitry in hESCs. This article is protected by copyright. All rights reserved

Human pluripotent stem cells, with their ability to proliferate indefinitely and to differentiate into virtually all cell types of the human body, provide a novel resource to study human development and to implement relevant disease models. Here, we employed a human pancreatic differentiation platform complemented with an shRNA screen in human pluripotent stem cells (PSCs) to identify potential drivers of early endoderm and pancreatic development. Deep sequencing followed by abundancy ranking pinpointed six top hit genes potentially associated with either improved or impaired endodermal differentiation, which were selected for functional validation in CRISPR-Cas9 mediated knockout (KO) lines. Upon endoderm differentiation (DE), particularly the loss of SLC22A1 and DSC2 led to impaired differentiation efficiency into CXCR4/KIT-positive DE cells. qPCR analysis also revealed changes in differentiation markers CXCR4, FOXA2, SOX17, and GATA6. Further differentiation of PSCs to the pancreatic progenitor (PP) stage resulted in a decreased proportion of PDX1/NKX6-1-positive cells in SLC22A1 KO lines, and in DSC2 KO lines when differentiated under specific culture conditions. Taken together, our study reveals novel genes with potential roles in early endodermal development., publishedVersion

Early in animal development many cells are conditionally specified based on observations that those cells can be directed toward alternate fates. The endomesoderm is so named because early specification produces cells that often have been observed to simultaneously express both early endoderm and mesoderm transcription factors. Experiments with these cells demonstrate that their progeny can directed entirely toward endoderm or mesoderm, whereas normally they establish both germ layers. This review examines the mechanisms that initiate the conditional endomesoderm state, its metastability, and the mechanisms that resolve that state into definitive endoderm and mesoderm.

Hypoblast/visceral endoderm assists in amniote nutrition, axial positioning and formation of the gut. Here, we provide evidence, currently limited to humans and non-human primates, that hypoblast is a purveyor of extraembryonic mesoderm in the mouse gastrula. Fate mapping a unique segment of axial extraembryonic visceral endoderm associated with the allantoic component of the primitive streak, and referred to as the "AX", revealed that visceral endoderm supplies the placentae with extraembryonic mesoderm. Exfoliation of the AX was dependent upon contact with the primitive streak, which modulated Hedgehog signaling. Resolution of the AX's epithelial-to-mesenchymal transition (EMT) by Hedgehog shaped the allantois into its characteristic projectile and individualized placental arterial vessels. A unique border cell separated the delaminating AX from the yolk sac blood islands which, situated beyond the limit of the streak, were not formed by an EMT. Over time, the AX became the hindgut lip, which contributed extensively to the posterior interface, including both embryonic and extraembryonic tissues. The AX, in turn, imparted antero-posterior (A-P) polarity on the primitive streak and promoted its elongation and differentiation into definitive endoderm. Results of heterotopic grafting supported mutually interactive functions of the AX and primitive streak, showing that together, they self-organized into a complete version of the fetal-placental interface, forming an elongated structure that exhibited A-P polarity and was composed of the allantois, an AX-derived rod-like axial extension reminiscent of the embryonic notochord, the placental arterial vasculature and visceral endoderm/hindgut.

β-catenin-mediated signaling has been extensively studied in regard to its role in the regulation of human embryonic stem cells (hESCs). However, the results are controversial and the mechanism by which β-catenin regulates the hESC fate remains unclear. Here, we report that β-catenin and γ-catenin are functionally redundant in mediating hESC adhesion and are required for embryoid body formation, but both genes are dispensable for hESC maintenance, as the undifferentiated state of β-catenin and γ-catenin double deficient hESCs can be maintained. Overexpression of β-catenin induces rapid hESC differentiation. Functional assays revealed that TCF1 plays a crucial role in hESC differentiation mediated by β-catenin. Forced expression of TCF1, but not other LEF1/TCF family members, resulted in hESC differentiation towards the definitive endoderm. Conversely, knockdown of TCF1 or inhibition of the interaction between TCF1 and β-catenin delayed hESC exit from pluripotency. Furthermore, we demonstrated that GATA6 plays a predominant role in TCF1-mediated hESC differentiation. Knockdown of GATA6 completely eliminated the effect of TCF1, while forced expression of GATA6 induced hESC differentiation. Our data thus reveal more detailed mechanisms for β-catenin in regulating hESC fate decisions and will expand our understanding of the self-renewal and differentiation circuitry in hESCs.

Objectives Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) can generate any given cell type in the human body. One challenge for cell-replacement therapy is the efficient differentiation and expansion of large quantities of progenitor cells from pluripotent stem cells produced under good manufacturing practice (GMP). FOXA2 and SOX17 double positive definitive endoderm (DE) progenitor cells can give rise to all endoderm-derived cell types in the thymus, thyroid, lung, pancreas, liver, and gastrointestinal tract. FOXA2 is a pioneer transcription factor in DE differentiation that is also expressed and functionally required during pancreas development and islet cell homeostasis. Current differentiation protocols can successfully generate endoderm; however, generation of mature glucose-sensitive and insulin-secreting β-cells is still a challenge. As a result, it is of utmost importance to screen for small molecules that can improve DE and islet cell differentiation for cell-replacement therapy for diabetic patients. Methods The aim of this study was to identify and validate small molecules that can induce DE differentiation and further enhance pancreatic progenitor differentiation. Therefore, we developed a large scale, high-content screen for testing a chemical library of 23,406 small molecules to identify compounds that induce FoxA2 in mouse embryonic stem cells (mESCs). Results Based on our high-content screen algorithm, we selected 84 compounds that directed differentiation of mESCs towards the FoxA2 lineage. Strikingly, we identified ROCK inhibition (ROCKi) as a novel mechanism of endoderm induction in mESCs and hESCs. DE induced by the ROCK inhibitor Fasudil efficiently gives rise to PDX1+ pancreatic progenitors from hESCs. Conclusion Taken together, DE induction by ROCKi can simplify and improve current endoderm and pancreatic differentiation protocols towards a GMP-grade cell product for β-cell replacement., Highlights • High content screen of 23,406 small molecules identifies novel definitive endoderm inducers Fasudil and RKI-1447 in mESCs. • Fasudil and RKI-1447 induce anterior definitive endoderm differentiation in mESCs and hESCs through ROCK inhibition. • Fasudil and RKI-1447 further differentiates the ADE cells into PDX1+ pancreatic progenitors.

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.

Background The use of a personalized liver organoid derived from human-induced pluripotent stem cells (HuiPSCs) is advancing the use of in vitro disease models for the design of specific, effective therapies for individuals. Collecting patient peripheral blood cells for HuiPSC generation is preferable because it is less invasive; however, the capability of blood cell-derived HuiPSCs for hepatic differentiation and liver organoid formation remains uncertain. Moreover, the currently available methods for liver organoid formation require a multistep process of cell differentiation or a combination of hepatic endodermal, endothelial and mesenchymal cells, which is a major hurdle for the application of personalized liver organoids in high-throughput testing of drug toxicity and safety. To demonstrate the capability of blood cell-derived HuiPSCs for liver organoid formation without support from endothelial and mesenchymal cells. Methods The peripheral blood-derived HuiPSCs first differentiated into hepatic endoderm (HE) in two-dimensional (2D) culture on Matrigel-coated plates under hypoxia for 10 days. The HE was then collected and cultured in 3D culture using 50% Matrigel under ambient oxygen. The maturation of hepatocytes was further induced by adding hepatocyte growth medium containing HGF and oncostatin M on top of the 3D culture and incubating the culture for an additional 12–17 days. The function of the liver organoids was assessed using expression analysis of hepatocyte-specific gene and proteins. Albumin (ALB) synthesis, glycogen and lipid storage, and metabolism of indocyanine were evaluated. The spatial distribution of albumin was examined using immunofluorescence and confocal microscopy. Results CD34+ hematopoietic cell-derived HuiPSCs were capable of differentiating into definitive endoderm expressing SOX17 and FOXA2, hepatic endoderm expressing FOXA2, hepatoblasts expressing AFP and hepatocytes expressing ALB. On day 25 of the 2D culture, cells expressed SOX17, FOXA2, AFP and ALB, indicating the presence of cellular heterogeneity. In contrast, the hepatic endoderm spontaneously formed a spherical, hollow structure in a 3D culture of 50% Matrigel, whereas hepatoblasts and hepatocytes could not form. Microscopic observation showed a single layer of polygonal-shaped cells arranged in a 3D structure. The hepatic endoderm-derived organoid synthesis ALB at a higher level than the 2D culture but did not express definitive endoderm-specific SOX17, indicating the greater maturity of the hepatocytes in the liver organoids. Confocal microscopic images and quantitative ELISA confirmed albumin synthesis in the cytoplasm of the liver organoid and its secretion. Overall, 3D culture of the hepatic endoderm is a relatively fast, simple, and less laborious way to generate liver organoids from HuiPSCs that is more physiologically relevant than 2D culture.

Summary Transcriptome analysis has uncovered a series of long noncoding RNAs (lncRNAs) transcribed during cell differentiation, but how lncRNA is integrated with known transcriptional regulatory network is poorly understood. Here, we utilize human definitive endoderm differentiation as a model system and decipher the functional interaction between lncRNA and key transcriptional factor. We have identified GATA6-AS1, an lncRNA divergently transcribed from the GATA6 locus, is highly expressed during endoderm differentiation. Knockdown of GATA6-AS1 in human pluripotent stem cells has no influence on morphology and pluripotency; however, GATA6-AS1 depletion causes the deficiency of definitive endoderm differentiation. GATA6-AS1 positively regulates the expression of endoderm key factor GATA6. Further investigation shows GATA6-AS1 interacts with SMAD2/3 and activates the transcription of GATA6. In addition, overexpression of GATA6 is able to rescue the defect of endoderm differentiation due to the absence of GATA6-AS1, suggesting that GATA6 is the functional target of GATA6-AS1 during endoderm differentiation. Ultimately, our study reveals that GATA6-AS1 is necessary for human endoderm specification and reveals the underlying mechanism between GATA6-AS1 and GATA6., Graphical Abstract, Highlights • GATA6-AS1 is a lncRNA highly expressed in human endoderm with two isoforms • GATA6-AS1 controls human endoderm differentiation through regulating GATA6 • GATA6-AS1 regulates GATA6, the functional target in endoderm differentiation • GATA6-AS1 is required for SMAD2/3-mediated GATA6 transcriptional activation, In this study, Jiang and colleagues have identified an endoderm-expressed lncRNA, GATA6-AS1, which is necessary for definitive endoderm differentiation. GATA6-AS1 deletion causes the differentiation deficiency of human definitive endoderm. Further mechanistic study shows that GATA6-AS1 interacts with SMAD2/3 and recruits SMAD2/3 to activate GATA6 transcription, thus contributing to endoderm differentiation.

In this article, we report for the first time the derivation and characterization of extra-embryonic endoderm (XEN) cells from primitive endoderm (PrE) of porcine (p) embryos. The pXEN cells can be reliably and reproducibly generated from parthenote, in vitro and in vivo derived embryos. The pXEN cells retained all the hallmarks of PrE including expression of canonical PrE and XEN cell markers (GATA4, GATA6, SOX17, SALL4, FOXA2, and HNF4A). Transcriptome analysis further confirmed their XEN cell origin. The pXEN cells when introduced into blastocyst stage embryo contributed to wide-spread chimerism including visceral yolk sac, chorion, as well as embryonic gut and liver primordium in the fetus. The pXEN cells were shown to be an efficient nuclear donor for generating cloned offspring. Taken together, pXEN cells fulfil a longstanding need for a stable, chimera-competent, and nuclear transfer-compatible porcine embryonic cells with applications for agriculture and medicine.Significance StatementWe report for the first time, the derivation and characterization of extraembryonic endoderm (XEN) stem cells from porcine (p) embryos. The pXEN cells can be reliably and reproducibly derived from primitive endoderm precursors. When injected into blastocyst-stage embryos, the pXEN cells have contributed to wide-spread chimerism including visceral yolk sac, chorion of the extraembryonic membranes, as well as definitive endoderm of the fetus, primarily the embryonic gut and liver primordium. Additionally, these XEN cells have proven to be an efficient nuclear donor for generating cloned offspring. These newly discovered stem cells provide a novel model for studying lineage segregation, as well as a source for interspecies chimeras for generating endodermal organs, and for genome editing in livestock.

Markers that distinguish pancreatic and hepatic progenitors improve stem-cell differentiation to pancreatic beta cells.Methods for differentiating human pluripotent stem cells to pancreatic and liver lineages in vitro have been limited by the inability to identify and isolate distinct endodermal subpopulations specific to these two organs. Here we report that pancreatic and hepatic progenitors can be isolated using the surface markers CD177/NB1 glycoprotein and inducible T-cell costimulatory ligand CD275/ICOSL, respectively, from seemingly homogeneous definitive endoderm derived from human pluripotent stem cells. Anterior definitive endoderm (ADE) subpopulations identified by CD177 and CD275 show inverse activation of canonical and noncanonical WNT signaling. CD177(+) ADE expresses and synthesizes the secreted WNT, NODAL and BMP antagonist CERBERUS1 and is specified toward the pancreatic fate. CD275(+) ADE receives canonical Wnt signaling and is specified toward the liver fate. Isolated CD177(+) ADE differentiates more homogeneously into pancreatic progenitors and into more functionally mature and glucose-responsive beta-like cells in vitro compared with cells from unsorted differentiation cultures.

Gastrulation is a central process in mammalian development in which a spatiotemporally coordinated series of events driven by cross-talk between adjacent embryonic and extra-embryonic tissues results in stereotypical morphogenetic cell behaviors, massive cell proliferation and the acquisition of distinct cell identities. Gastrulation provides the blueprint of the body plan of the embryo, as well as generating extra-embryonic cell types of the embryo to make a connection with its mother. Gastrulation involves the specification of mesoderm and definitive endoderm from pluripotent epiblast, concomitant with a highly ordered elongation of tissue along the anterior-posterior (AP) axis. Interestingly, cells with an endoderm identity arise twice during mouse development. Cells with a primitive endoderm identity are specified in the preimplantation blastocyst, and which at gastrulation intercalate with the emergent definitive endoderm to form a mosaic tissue, referred to as the gut endoderm. The gut endoderm gives rise to the gut tube, which will subsequently become patterned along its AP axis into domains possessing unique visceral organ identities, such as thyroid, lung, liver and pancreas. In this way, proper endoderm development is essential for vital organismal functions, including the absorption of nutrients, gas exchange, detoxification and glucose homeostasis.

Embryonic development is widely studied due to its application in disease treatment. The published literature demonstrated that Kruppel-like factor 8(KLF8) plays an important role in modulating mesendoderm to definitive endoderm (DE) differentiation. However, it is not clear how KLF8 interacts with other key genes and affects the differentiation process. To qualitatively and quantitatively explore the molecular mechanisms of KLF8 during the differentiation of human embryonic stem cells (hESCs) in detail, we developed a mathematical model to describe the dynamics between KLF8 and two other significant genes, E-cadherin(CDH1) and Zinc-finger E-box-binding homeobox1(ZEB1). Based on the single-cell RNA-seq data, the model structure and parameters were obtained using particle swarm optimization (PSO). The bifurcation analysis and simulation results reveal that the system can exhibit a complex tristable transition, which corresponds to the three states of embryonic development at the single-cell level. We further predict that the novel important gene KLF8 promotes the formation of DE cells by reciprocal inhibition between CDH1 and KLF8 and promotion of the expression of ZEB1. These results may help to shed light on the biological mechanism in the differentiation process of hESCs.

The segregation of definitive endoderm (DE) from mesendoderm progenitors leads to the formation of two distinct germ layers. Dissecting DE onset has been challenging as it occurs within a narrow spatio-temporal window in the embryo. Here we employ a dual Bra-GFP, Sox17-RFP reporter cell line to study DE onset dynamics. We find Sox17 starts in a few isolated cellsin vivo. Using 2D and 3Din vitromodels, we show that DE cells emerge from mesendoderm progenitors at a temporally regular, but spatially stochastic pattern, which is subsequently arranged by self-sorting of Sox17+ cells. Self-sorting coincides with up-regulation of E-cadherin but is not necessary for DE differentiation or proliferation. A subpopulation of Bra-high cells commits to a Sox17+ fate independent of external Wnt signal. Ourin vivoandin vitroresults highlight basic rules governing DE onset and patterning through the commonalities and differences between these systems.

Human induced pluripotent stem cells (hiPSCs) not only offer great opportunities for the study of human development but also have tremendous potential for future clinical cell-based therapies. The protocol outlined here is used to differentiate hiPSCs into lung epithelial cell types through a process that faithfully recapitulates the stepwise events observed in vivo. From pluripotency, cells are differentiated to a definitive endoderm fate, followed by progression into anteriorized foregut endoderm that has the ability to give rise to both proximal and distal epithelial cells. Furthermore, this methodology allows for the study of lung dysfunction and disease modeling using patient-derived cells, as well as high-throughput pharmacological screening and eventually personalized therapies. Recently we were able to reproduce this protocol using the working cell bank of an hiPSC line made under current Good Manufacturing Practice (cGMP) conditions, a necessary step for the future clinical application of these cells. © 2019 by John Wiley & Sons, Inc.

Embryonic stem (ES) cells have a pluripotent ability to differentiate into a variety of cell lineages in vitro. Investigating the roles of the co-culture of mouse embryonic stem cells (mESCs) with pancreatic islets (PL) and liver stromal cells (LSCs) in the differentiation of definitive endoderm (DE) cells was the purpose of this study. Here by using PL derived from adult mouse and LSCs derived from mouse fetal liver, we are trying to introduce a new protocol which is devoid of growth factors. We calculated mESCs indirectly for 7 days and then we analyzed the resulting cells regarding DE genes and protein expression using qRT-PCR and immunocytochemistry. It was proved that mESCs can differentiate into DE cells and also co-culture system with PL provides a proper environment for differentiation of mESCs. However co-culture system with LSCs isolated from mouse fetal liver, are not able to increase DE differentiation. As the first report we showed that a PL induced microenvironment can improve DE differentiation. More modifications of the PL microenvironment may present another approach to providing DE cells for differentiation into hepatocyte and beta cells. By applying these results, production of DE from stem cells facilitates in vitro.

Definitive endoderm is the cellular precursor to respiratory- and digestive-related organs such as lungs, stomach, liver, pancreas and intestine. Endodermal lineage cells derived from pluripotent stem cells (PSCs) in vitro are a potentially unlimited resource for regenerative medicine. These cells are useful tools for studying the physiology, pathogenesis and medical therapies involving these tissues, and great progress has been achieved in PSCs differentiation protocols. In this review, we will focus on the most common and/or advanced differentiation strategies currently used in generating endodermal lineage cells from PSCs. A brief discussion about the effect of early definitive endoderm differentiation on the final development products will follow.

Hepatocyte nuclear factor 4 alpha (HNF4α) is a key transcription factor for liver development. Although HNF4α is necessary for hepatoblast differentiation, the function of HNF4α before the hepatoblast differentiation, such as in definitive endoderm differentiation, is not well known. In addition, it is known that there are nine HNF4α isoforms, but the expression and function of each HNF4α isoform during the definitive endoderm differentiation is also not clear. In this study, we examined the expression pattern of HNF4α and its functions in the definitive endoderm differentiation from human induced pluripotent stem (iPS) cells. We found that the HNF4α-1D isoform expression levels were significantly increased during the definitive endoderm differentiation, while the HNF4α-1A isoform expression levels did not change. Therefore, we further examined the function of the HNF4α-1D isoform in definitive endoderm differentiation. HNF4α-1D overexpression or knockdown was found to promote or prevent the definitive endoderm differentiation, respectively. Interestingly, Lefty1 was directly regulated by HNF4α-1D, and Lefty1 knockdown also prevented the definitive endoderm differentiation. These results suggest that HNF4α-1D promotes definitive endoderm differentiation through the regulation of Lefty1. To our knowledge, this is the first report to clarify the expression pattern and function of HNF4α during the definitive endoderm differentiation.

Many reports have described methods that induce definitive endoderm (DE) cells from human pluripotent stem cells (hPSCs). However, it is unclear whether the differentiation propensity of these DE cells is uniform. This uncertainty is due to the different developmental stages that give rise to anterior and posterior DE from anterior primitive streak (APS). Therefore, these DE cell populations might be generated from the different stages of APS cells, which affect the DE cell differentiation potential. Here, we succeeded in selectively differentiating early and late APS cells from human induced pluripotent stem cells (hiPSCs) using different concentrations of CHIR99021, a small molecule Wnt/β-catenin pathway activator. We also established novel differentiation systems from hiPSCs into three types of DE cells: anterior and posterior domains of anterior DE cells through early APS cells and posterior DE cells through late APS cells. These different DE cell populations could differentiate into distinct endodermal lineages in vitro, such as lung, liver or small intestine progenitors. These results indicate that different APS cells can produce distinct types of DE cells that have proper developmental potency and suggest a method to evaluate the quality of endodermal cell induction from hPSCs.