Definitive Endoderm Differentiation Research Articles

Regeneration of insulin-producing cells from iPS cells using functionalized scaffolds and solid lipid nanoparticles

BackgroundFrom ongoing human clinical trials, cell therapy for diabetic treatment using islet-like organoid generation from induced pluripotent stem cells (iPSCs) appears to have a promising future. MethodsDevelopment of islet cells from iPSCs was guided in activin A- and Wnt3a-grafted gelatin-polyacrylamide inverted colloidal crystal scaffolds (activin A-Wnt3a-Gel-PAAM ICCS) with pore sizes of 70 and 160 µm, followed by regulation with fibroblast growth factor 7-grafted retinoic acid-entrapped solid lipid nanoparticles (FGF7-RA-SLNs). Significant FindingsFGF7-RA-SLNs diffused into activin A-Wnt3a-Gel-PAAM ICCS, and controlled differentiation of definitive endoderm (DE) cells into islet cells. The immunochemical staining, flow cytometry and western blot demonstrated combined effects among activin A and Wnt3a, FGF7 and RA in materials for the 2-step differentiation. Wnt3a on the pore surface promoted activin A activity for generating DE cells, and FGF7 enhanced RA activity for producing islet cells. Under glucose stimulation, activin A-Wnt3a-Gel-PAAM ICCS with pore sizes of 70 µm were prone to regenerate insulin-producing β-cells rather than α cells. The optimized conditions for insulin secretion from β-like cells in this study were pore size of 70 µm, Gel:PAAM of 7:3, 100 ng/mL activin A, 100 ng/mL Wnt3a, 50 ng/mL FGF7 and 600 ng/mL RA. Activin A-Wnt3a-Gel-PAAM ICCS associated with FGF7-RA-SLNs can be effective in forming biomimetic pancreas and supporting cell therapy from iPSCs for diabetic management.

Mitochondrial homeostasis regulates definitive endoderm differentiation of human pluripotent stem cells

Cellular organelles play fundamental roles in almost all cell behaviors. Mitochondria have been reported to be functionally linked to various biological processes, including reprogramming and pluripotency maintenance. However, very little about the role of mitochondria has been revealed in human early development and lineage specification. Here, we reported the characteristics and function of mitochondria during human definitive endoderm differentiation. Using a well-established differentiation system, we first investigated the change of mitochondrial morphology by comparing undifferentiated pluripotent stem cells, the intermediate mesendoderm cells, and differentiated endoderm cells, and found that mitochondria were gradually elongated and matured along differentiation. We further analyzed the expression pattern of mitochondria-related genes by RNA-seq, indicating that mitochondria became active during differentiation. Supporting this notion, the production of adenosine triphosphate (ATP) and reactive oxygen species (ROS) was increased as well. Functionally, we utilized chemicals and genome editing techniques, which could interfere with mitochondrial homeostasis, to determine the role of mitochondria in human endoderm differentiation. Treatment with mitochondrial inhibitors, or genetic depletion of mitochondrial transcription factor A (TFAM), significantly reduced the differentiation efficiency of definitive endoderm. In addition, the defect in endoderm differentiation due to dysfunctional mitochondria could be restored to some extent by the addition of ATP. Moreover, the clearance of excessive ROS due to dysfunctional mitochondria by N-acetylcysteine (NAC) improved the differentiation as well. We further found that ATP and NAC could partially replace the growth factor activin A for definitive endoderm differentiation. Our study illustrates the essential role of mitochondria during human endoderm differentiation through providing ATP and regulating ROS levels, which may provide new insight for metabolic regulation of cell fate determination.

Parallel functional assessment of m6A sites in human endodermal differentiation with base editor screens

N6-methyladenosine (m6A) plays important role in lineage specifications of embryonic stem cells. However, it is still difficult to systematically dissect the specific m6A sites that are essential for early lineage differentiation. Here, we develop an adenine base editor-based strategy to systematically identify functional m6A sites that control lineage decisions of human embryonic stem cells. We design 7999 sgRNAs targeting 6048 m6A sites to screen for m6A sites that act as either boosters or barriers to definitive endoderm specification of human embryonic stem cells. We identify 78 sgRNAs enriched in the non-definitive endoderm cells and 137 sgRNAs enriched in the definitive endoderm cells. We successfully validate two definitive endoderm promoting m6A sites on SOX2 and SDHAF1 as well as a definitive endoderm inhibiting m6A site on ADM. Our study provides a functional screening of m6A sites and paves the way for functional studies of m6A at individual m6A site level.

Toward Xeno-Free Differentiation of Human Induced Pluripotent Stem Cell-Derived Small Intestinal Epithelial Cells

The small intestinal epithelium has an important role in nutrition, but also in drug absorption and metabolism. There are a few two-dimensional (2D) patient-derived induced pluripotent stem cell (iPSC)-based intestinal models enabling easy evaluation of transcellular transport. It is known that animal-derived components induce variation in the experimental outcomes. Therefore, we aimed to refine the differentiation protocol by using animal-free components. More specifically, we compared maturation of 2D-cultured iPCSs toward small intestinal epithelial cells when cultured either with or without serum, and either on Geltrex or on animal-free, recombinant laminin-based substrata. Differentiation status was characterized by qPCR, immunofluorescence imaging, and functionality assays. Our data suggest that differentiation toward definitive endoderm is more efficient without serum. Both collagen- and recombinant laminin-based coating supported differentiation of definitive endoderm, posterior definitive endoderm, and small intestinal epithelial cells from iPS-cells equally well. Small intestinal epithelial cells differentiated on recombinant laminin exhibited slightly more enterocyte specific cellular functionality than cells differentiated on Geltrex. Our data suggest that functional small intestinal epithelial cells can be generated from iPSCs in serum-free method on xeno-free substrata. This method is easily converted to an entirely xeno-free method.

Emergence and patterning dynamics of mouse-definitive endoderm

SummaryThe segregation of definitive endoderm (DE) from bipotent 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 spatiotemporal window in the embryo. Here, we employ a dual Bra/Sox17 reporter cell line to study DE onset dynamics. We find Sox17 expression initiates in vivo in isolated cells within a temporally restricted window. In 2D and 3D in vitro models, 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 upregulation 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.

MiR-27b antagonizes BMP signaling in early differentiation of human induced pluripotent stem cells

Human induced pluripotent stem (hiPS) cells are feasible materials for studying the biological mechanisms underlying human embryogenesis. In early embryogenesis, definitive endoderm and mesoderm are differentiated from their common precursor, mesendoderm. Bone morphogenetic protein (BMP) signaling is responsible for regulating mesendoderm and mesoderm formation. Micro RNAs (miRNAs), short non-coding RNAs, broadly regulate biological processes via post-transcriptional repression. The expression of miR-27b, which is enriched in somatic cells, has been reported to increase through definitive endoderm and hepatic differentiation, but little is known about how miR-27b acts during early differentiation. Here, we used miR-27b-inducible hiPS cells to investigate the roles of miR-27b in the undifferentiated and early-differentiated stages. In undifferentiated hiPS cells, miR-27b suppressed the expression of pluripotency markers [alkaline phosphatase (AP) and nanog homeobox (NANOG)] and cell proliferation. Once differentiation began, miR-27b expression repressed phosphorylated SMAD1/5, the mediators of the BMP signaling, throughout definitive endoderm differentiation. Consistent with the above findings, miR-27b overexpression downregulated BMP-induced mesendodermal marker genes [Brachyury, mix paired-like homeobox 1 (MIXL1) and eomesodermin (EOMES)], suggesting that miR-27b had an inhibitory effect on early differentiation. Collectively, our findings revealed a novel antagonistic role of miR-27b in the BMP signaling pathway in the early differentiation of hiPS cells.

Generation of pancreatic progenitors from human pluripotent stem cells by small molecules.

SummaryHuman 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.

Emergence and Patterning Dynamics of Mouse Definitive Endoderm

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.

Involvement of LIMK2 in actin cytoskeleton remodeling during the definitive endoderm differentiation

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.

Automated optimization of endoderm differentiation on chip.

Human induced pluripotent stem cells (hiPSCs) can serve as an unlimited source to rebuild organotypic tissues in vitro. Successful engineering of functional cell types and complex organ structures outside the human body requires knowledge of the chemical, temporal, and spatial microenvironment of their in vivo counterparts. Despite an increased understanding of mouse and human embryonic development, screening approaches are still required for the optimization of stem cell differentiation protocols to gain more functional mature cell types. The liver, lung, pancreas, and digestive tract originate from the endoderm germ layer. Optimization and specification of the earliest differentiation step, which is the definitive endoderm (DE), is of central importance for generating cell types of these organs because off-target cell types will propagate during month-long cultivation steps and reduce yields. Here, we developed a microfluidic large-scale integration (mLSI) chip platform for combined automated three-dimensional (3D) cell culturing and high-throughput imaging to investigate anterior/posterior patterns occurring during hiPSC differentiation into DE cells. Integration of 3D cell cultures with a diameter of 150 μm was achieved using a U-shaped pneumatic membrane valve, which was geometrically optimized and fluidically characterized. Upon parallelization of 32 fluidically individually addressable cell culture unit cells with a total of 128 3D cell cultures, complex and long-term DE differentiation protocols could be automated. Real-time bright-field imaging was used to analyze cell growth during DE differentiation, and immunofluorescence imaging on optically cleared 3D cell cultures was used to determine the DE differentiation yield. By systematically alternating transforming growth factor β (TGF-β) and WNT signaling agonist concentrations and temporal stimulation, we showed that even under similar DE differentiation yields, there were patterning differences in the 3D cell cultures, indicating possible differentiation differences between established DE protocols. The automated mLSI chip platform with the general analytical workflow for 3D stem cell cultures offers the optimization of in vitro generation of various cell types for cell replacement therapies.

Non-xenogeneic expansion and definitive endoderm differentiation of human pluripotent stem cells in an automated bioreactor.

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 and induced pluripotent stem cellsto 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 an SSB with controlled pH and dissolved oxygen. Cultures displayed a 10- to 12-fold increase in cell number over 5-6 days with the 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 study advances bioprocess development for producing a wide gamut of human DE cell-derived therapeutics.

Dynamic Click Hydrogels for Xeno-Free Culture of Induced Pluripotent Stem Cells.

Xeno-free, chemically defined poly(ethylene glycol) (PEG)-based hydrogels are being increasingly used for in vitro culture and differentiation of human induced pluripotent stem cells (hiPSCs). These synthetic matrices provide tunable gelation and adaptable material properties crucial for guiding stem cell fate. Here, sequential norbornene-click chemistries are integrated to form synthetic, dynamically tunable PEG-peptide hydrogels for hiPSCs culture and differentiation. Specifically, hiPSCs are photoencapsulated in thiol-norbornene hydrogels crosslinked by multiarm PEG-norbornene (PEG-NB) and proteaselabile crosslinkers. These matrices are used to evaluate hiPSC growth under the influence of extracellular matrix properties. Tetrazine-norbornene (Tz-NB) click reaction is then employed to dynamically stiffen the cell-laden hydrogels. Fast reactive Tz and its stable derivative methyltetrazine (mTz) are tethered to multiarm PEG, yielding mono-functionalized PEG-Tz, PEG-mTz, and dualfunctionalized PEG-Tz/mTz that react with PEG-NB to form additional crosslinks in the cell-laden hydrogels. The versatility of Tz-NB stiffening is demonstrated with different Tz-modified macromers or by intermittent incubation of PEG-Tz for temporal stiffening. Finally, the Tz-NB-mediated dynamic stiffening is explored for 4D culture and definitive endoderm differentiation of hiPSCs. Overall, this dynamic hydrogel platform affords exquisite controls of hydrogel crosslinking for serving as a xeno-free and dynamic stem cell niche.

Enhanced Induction of Definitive Endoderm Differentiation of Mouse Embryonic Stem Cells in Simulated Microgravity.

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 (Oct3/4) and mesendodermal (Brachyury T), endodermal (FoxA2, Sox17, CxCr4), and mesodermal (Vimentin, Meox1) lineages. Culture in the form of EBs in maintenance media in the presence of leukemia inhibitory factor, in static or SMG conditions, induced expression of some of the differentiation markers, suggesting heterogeneity of the cells. This is in line with previous studies showing that differentiation is initiated as cells are aggregated into EBs even without supplementing differentiation factors to the media. Culturing EBs in static conditions in differentiation media (DM) in the presence of activin A reduced Oct3/4 expression and significantly increased Brachyury T and CxCr4 expression, but downregulated FoxA2 and Sox17. However, culturing in SMG BRs in DM upregulated Brachyury T and all of the DE markers and reduced Oct3/4 expression, indicating the advantage of dynamic cultures in BRs to specifically enhance directed DE differentiation. Given the potential discrepancies between the SMG conditions on earth and actual microgravity conditions, as observed in other studies, future experiments in space flight are required to validate the effects of reduced gravity on mESC differentiation.

GATA6-AS1 Regulates GATA6 Expression to Modulate Human Endoderm Differentiation.

SummaryTranscriptome 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.

The Role of SMAD2/3 in Human Embryonic Stem Cells.

Human embryonic stem cells (hESCs) possess the potential of long-term self-renewal and three primary germ layers differentiation, and thus hESCs are expected to have broad applications in cell therapy, drug screening and basic research on human early embryonic development. Many efforts have been put to dissect the regulation of pluripotency and direct differentiation of hESCs. TGFβ/Activin/Nodal signal pathway critically regulates pluripotency maintenance and cell differentiation through the main signal transducer SMAD2/3 in hESCs, but the action manners of SMAD2/3 in hESCs are sophisticated and not documented yet. Here we review and discuss the roles of SMAD2/3 in hESC pluripotency maintenance and differentiation initiation separately. We summarize that SMAD2/3 regulates pluripotency and differentiation mainly through four aspects, (1) controlling divergent transcriptional networks of pluripotency and differentiation; (2) interacting with chromatin modifiers to make the chromatin accessible or recruiting METTL3-METTL14-WTAP complex and depositing m6A to the mRNA of pluripotency genes; (3) acting as a transcription factor to activate endoderm-specific genes to thus initiate definitive endoderm differentiation, which happens as cyclin D/CDK4/6 downstream target in later G1 phase as well; (4) interacting with endoderm specific lncRNAs to promote differentiation.

Regulation of hepatic differentiation of human embryonic stem cells by calcium silicate extracts for liver injury repairing

Directing embryonic stem cell (ESC)-derived hepatocytes is critical in understanding hepatic differentiation and applying cell-based treatment to severe liver diseases. While growth factor-based strategies are widely used, using chemical cues could present an alternative to optimize the strategies for stem cell differentiation. Here, for the first time, an inorganic calcium silicate (CS, CaSiO3)-based approach, together with a modified four-stage differentiating protocol, was proposed to quantify the effects of CS extracts on inducing hepatic differentiation of human ESCs (H9 cells). The roles of CS-activated H9 cells in liver injury repair were tested by cell tracking and immunohistochemical staining. Results indicated that high concentrations of CS extracts initially enhance definitive endodermal (DE) lineage, followed by gradual DE differentiation at low CS concentrations. The order of CS addition is also crucial, since the presence at stemness stage and the absence at DE stage could optimize hepatic differentiation capacity of H9 cells, resulting in optimized cells that differentiate into functional hepatocyte-like cells. The addition of CS extracts at precursor hepatocyte stage enhances their maturity, which favors the turnover of liver injury in CCl4-treated mice. These results provide an insight into applying bioactive inorganic biomaterials to foster hepatic differentiation of human ESCs for cell therapy.

TGFβ-dependent mitochondrial biogenesis is activated during definitive endoderm differentiation.

Whether mitochondrial remodeling and metabolic reprogramming occur during the differentiation of human embryonic stem cells (hESCs) to definitive endoderm (DE) is unknown. We found that fragmented and punctate mitochondria in undifferentiated hESCs progressively fused into an extensive and branched network upon DE differentiation. Mitochondrial mass and mitochondrial DNA (mtDNA) content were significantly increased with the upregulated expression of mitochondrial biogenesis regulator PGC1-A upon DE differentiation, accompanied by the rise of the amount of ATP (2.5-fold) and its by-product reactive oxygen species (2.0-fold). We observed that in contrast to a shutoff of glycolysis, expressions of oxidative phosphorylation (OXPHOS) genes were increased, indicating that a transition from glycolysis to OXPHOS was tightly coupled to DE differentiation. In the meantime, we discovered that inhibition of TGF-β signaling led to impaired mitochondrial biogenesis and disturbed metabolic switch upon DE differentiation. Our work, for the first time, reports that TGF-β signaling-dependent mitochondrial biogenesis and metabolic reprogramming occur during early endodermal differentiation.

FGF signaling mediates definitive endoderm formation by regulating epithelial-to-mesenchymal transition and cell proliferation.

FGF signaling pathway is imperative for definitive endoderm (DE) differentiation from human embryonic stem cells (hESCs), which always accompanies an epithelial-to-mesenchymal transition (EMT) process. However, whether there is an association between FGF signaling and the EMT during DE formation in vitro has remained elusive. In the present study, we identify that several FGF family members were significantly activated during the differentiation of hESCs toward DE. Inhibition of FGF signaling by an efficient and selective inhibitor BGJ398 abolishes both the EMT and DE induction by blocking the activation of the zinc-finger transcription factor SNAI1 which is a direct transcriptional repressor of cell adhesion protein CDH1. In addition, cell proliferation is also severely influenced by attenuating the FGF signaling. Collectively, we propose that the FGF signaling promotes the DE formation through mediating the EMT and cell proliferation.

Chemically-Defined, Xeno-Free, Scalable Production of hPSC-Derived Definitive Endoderm Aggregates with Multi-Lineage Differentiation Potential.

For the production and bio-banking of differentiated derivatives from human pluripotent stem cells (hPSCs) in large quantities for drug screening and cellular therapies, well-defined and robust procedures for differentiation and cryopreservation are required. Definitive endoderm (DE) gives rise to respiratory and digestive epithelium, as well as thyroid, thymus, liver, and pancreas. Here, we present a scalable, universal process for the generation of DE from human-induced pluripotent stem cells (hiPSCs) and embryonic stem cells (hESCs). Optimal control during the differentiation process was attained in chemically-defined and xeno-free suspension culture, and high flexibility of the workflow was achieved by the introduction of an efficient cryopreservation step at the end of DE differentiation. DE aggregates were capable of differentiating into hepatic-like, pancreatic, intestinal, and lung progenitor cells. Scale-up of the differentiation process using stirred-tank bioreactors enabled production of large quantities of DE aggregates. This process provides a useful advance for versatile applications of DE lineages, in particular for cell therapies and drug screening.

Single-cell qPCR demonstrates that Repsox treatment changes cell fate from endoderm to neuroectoderm and disrupts epithelial-mesenchymal transition.

A definitive endodermal cell lineage is a prerequisite for the efficient generation of mature endoderm derivatives that give rise to organs, such as the pancreas and liver. We previously reported that the induction of mesenchymal definitive endoderm cells depends on autocrine TGF-β signaling and that pharmacological blockage of TGF-β signaling by Repsox disrupts endoderm specification. The definitive endoderm arises from a primitive streak, which depends largely on TGF-β signaling. If the TGF-β pathway is blocked by Repsox, cell fate after the primitive streak induction is so-far unknown. We report here, that an induced primitive streak cell-population contained many T/SOX2 co-expressing cells, and subsequent inhibition of TGF-β signaling by Repsox promoted neuroectodermal cell fate, which was characterized using single-cell qPCR analysis and immunostaining. The process of epithelial-to-mesenchymal transition, which is inherent to the process of definitive endoderm differentiation, was also disrupted upon Repsox treatment. Our findings may provide a new approach to produce neural progenitors.