The fork protection complex promotes parental histone recycling and epigenetic memory.

The inheritance of parental histones across the replication fork is thought to mediate epigenetic memory. Here, we reveal that fission yeast Mrc1 (CLASPIN in humans) binds H3-H4 tetramers and operates as a central coordinator of symmetric parental histone inheritance. Mrc1 mutants in a key connector domain disrupted segregation of parental histones to the lagging strand comparable to Mcm2 histone-binding mutants. Both mutants showed clonal and asymmetric loss of H3K9me-mediated gene silencing. AlphaFold predicted co-chaperoning of H3-H4 tetramers by Mrc1 and Mcm2, with the Mrc1 connector domain bridging histone and Mcm2 binding. Biochemical and functional analysis validated this model and revealed a duality in Mrc1 function: disabling histone binding in the connector domain disrupted lagging-strand recycling while another histone-binding mutation impaired leading strand recycling. We propose that Mrc1 toggles histones between the lagging and leading strand recycling pathways, in part by intra-replisome co-chaperoning, to ensure epigenetic transmission to both daughter cells.

ERK signalling eliminates Nanog and maintains Oct4 to drive the formative pluripotency transition.

Naïve epiblast cells in the embryo and pluripotent stem cells in vitro undergo developmental progression to a formative state competent for lineage specification. During this transition, transcription factors and chromatin are rewired to encode new functional features. Here, we examine the role of mitogen-activated protein kinase (ERK1/2) signalling in pluripotent state transition. We show that a primary consequence of ERK activation in mouse embryonic stem cells is elimination of Nanog, which precipitates breakdown of the naïve state gene regulatory network. Variability in pERK dynamics results in heterogeneous loss of Nanog and metachronous state transition. Knockdown of Nanog allows exit without ERK activation. However, transition to formative pluripotency does not proceed and cells collapse to an indeterminate identity. This outcome is due to failure to maintain expression of the central pluripotency factor Oct4. Thus, during formative transition ERK signalling both dismantles the naïve state and preserves pluripotency. These results illustrate how a single signalling pathway can both initiate and secure transition between cell states.

SIRT1-driven mechanism: sevoflurane's interference with mESC neural differentiation via PRRX1/DRD2 cascade.

Investigating the sevoflurane-induced perturbation in the differentiation of mouse embryonic stem cells (mESCs) into neural stem cells (mNSCs), our study delineates a novel SIRT1/PRRX1/DRD2/PKM2/NRF2 axis as a key player in this intricate process. Sevoflurane treatment hindered mESC differentiation, evidenced by altered expression patterns of pluripotency and neural lineage markers. Mechanistically, sevoflurane downregulated Sirt1, setting in motion a signaling cascade. Sevoflurane may inhibit PKM2 dimerization and NRF2 signaling pathway activation by inhibiting the expression of SIRT1 and its downstream genes Prrx1 and DRD2, ultimately inhibiting mESCs differentiation into mNSCs. These findings contribute to our understanding of the molecular basis of sevoflurane-induced neural toxicity, presenting a potential avenue for therapeutic intervention in sevoflurane-induced perturbation in the differentiation of mESCs into mNSCs by modulating the SIRT1/PRRX1/DRD2/PKM2/NRF2 axis.

Intermittent ERK oscillations downstream of FGF in mouse embryonic stem cells.

Signal transduction networks generate characteristic dynamic activities to process extracellular signals and guide cell fate decisions such as to divide or differentiate. The differentiation of pluripotent cells is controlled by FGF/ERK signaling. However, only a few studies have addressed the dynamic activity of the FGF/ERK signaling network in pluripotent cells at high time resolution. Here, we use live cell sensors in wild-type and Fgf4-mutant mouse embryonic stem cells to measure dynamic ERK activity in single cells, for defined ligand concentrations and differentiation states. These sensors reveal pulses of ERK activity. Pulsing patterns are heterogeneous between individual cells. Consecutive pulse sequences occur more frequently than expected from simple stochastic models. Sequences become more prevalent with higher ligand concentration, but are rarer in more differentiated cells. Our results suggest that FGF/ERK signaling operates in the vicinity of a transition point between oscillatory and non-oscillatory dynamics in embryonic stem cells. The resulting heterogeneous dynamic signaling activities add a new dimension to cellular heterogeneity that may be linked to divergent fate decisions in stem cell cultures.

SETD3 regulates endoderm differentiation of mouse embryonic stem cells through canonical Wnt signaling pathway.

With self-renewal and pluripotency features, embryonic stem cells (ESCs) provide an invaluable tool to investigate early cell fate decisions. Pluripotency exit and lineage commitment depend on precise regulation of gene expression that requires coordination between transcription (TF) and chromatin factors in response to various signaling pathways. SET domain-containing 3 (SETD3) is a methyltransferase that can modify histones in the nucleus and actin in the cytoplasm. Through an shRNA screen, we previously identified SETD3 as an important factor in the meso/endodermal lineage commitment of mouse ESCs (mESC). In this study, we identified SETD3-dependent transcriptomic changes during endoderm differentiation of mESCs using time-course RNA-seq analysis. We found that SETD3 is involved in the timely activation of the endoderm-related gene network. The canonical Wnt signaling pathway was one of the markedly altered signaling pathways in the absence of SETD3. The assessment of Wnt transcriptional activity revealed a significant reduction in Setd3-deleted (setd3∆) mESCs coincident with a decrease in the nuclear pool of the key TF ß-catenin level, though no change was observed in its mRNA or total protein level. Furthermore, a proximity ligation assay (PLA) found an interaction between SETD3 and ß-catenin. We were able to rescue the differentiation defect by stably re-expressing SETD3 or activating the canonical Wnt signaling pathway by changing mESC culture conditions. Our results suggest that alterations in the canonical Wnt pathway activity and subcellular localization of ß-catenin might contribute to the endoderm differentiation defect of setd3∆ mESCs.

Cdk8 and Hira mutations trigger X chromosome elimination in naive female hybrid mouse embryonic stem cells.

Mouse embryonic stem cells (ESCs) possess a pluripotent developmental potential and a stable karyotype. An exception is the frequent loss of one X chromosome in female ESCs derived from inbred mice. In contrast, female ESCs from crosses between different Mus musculus subspecies often maintain two X chromosomes and can model X chromosome inactivation. Here we report that combined mutations of Hira and Cdk8 induce rapid loss of one X chromosome in a Mus musculus castaneus hybrid female ESC line that originally maintains two X chromosomes. We show that MEK1 inhibition, which is used for culturing naive pluripotent ESCs is sufficient to induce X chromosome loss. In conventional ESC media, Hira and Cdk8 mutant ESCs maintain both X chromosomes. Induction of X chromosome loss by switching to naive culture media allows us to perform kinetic measurements for calculating the chromosome loss rate. Our analysis shows that X chromosome loss is not explained by selection of XO cells, but likely driven by a process of chromosome elimination. We show that elimination of the X chromosome occurs with a rate of 0.3% per cell per division, which exceeds reported autosomal loss rates by 3 orders of magnitude. We show that chromosomes 8 and 11 are stably maintained. Notably, Xist expression from one of the two X chromosomes rescues X chromosomal instability in ΔHiraΔCdk8 ESCs. Our study defines mutations of Hira and Cdk8 as molecular drivers for X chromosome elimination in naive female ESCs and describes a cell system for elucidating the underlying mechanism.

B1 lymphocytes develop independently of Notch signaling during mouse embryonic development.

B1 lymphocytes are a small but unique component of the innate immune-like cells. However, their ontogenic origin is still a matter of debate. Although it is widely accepted that B1 cells originate early in fetal life, whether or not they arise from hematopoietic stem cells (HSCs) is still unclear. In order to shed light on the B1 cell origin, we set out to determine whether their lineage specification is dependent on Notch signaling, which is essential for the HSC generation and, therefore, all derivatives lineages. Using mouse embryonic stem cells (mESCs) to recapitulate murine embryonic development, we have studied the requirement for Notch signaling during the earliest B-cell lymphopoiesis and found that Rbpj-deficient mESCs are able to generate B1 cells. Their Notch independence was confirmed in ex vivo experiments using Rbpj-deficient embryos. In addition, we found that upregulation of Notch signaling induced the emergence of B2 lymphoid cells. Taken together, these findings indicate that control of Notch signaling dose is crucial for different B-cell lineage specification from endothelial cells and provides pivotal information for their in vitro generation from PSCs for therapeutic applications. This article has an associated 'The people behind the papers' interview.

Global and precise identification of functional miRNA targets in mESCs by integrative analysis.

MicroRNA (miRNA) loaded Argonaute (AGO) complexes regulate gene expression via direct base pairing with their mRNA targets. Previous works suggest that up to 60% of mammalian transcripts might be subject to miRNA-mediated regulation, but it remains largely unknown which fraction of these interactions are functional in a specific cellular context. Here, we integrate transcriptome data from a set of miRNA-depleted mouse embryonic stem cell (mESC) lines with published miRNA interaction predictions and AGO-binding profiles. Using this integrative approach, combined with molecular validation data, we present evidence that < 10% of expressed genes are functionally and directly regulated by miRNAs in mESCs. In addition, analyses of the stem cell-specific miR-290-295 cluster target genes identify TFAP4 as an important transcription factor for early development. The extensive datasets developed in this study will support the development of improved predictive models for miRNA-mRNA functional interactions.

Rapid quantitative high-throughput mouse embryoid body model for embryotoxicity assessment.

Individuals are exposed to a wide arrays of hazardous chemicals on a daily basis through various routes, many of which have not undergone comprehensive toxicity assessments. While traditional developmental toxicity tests involving pregnant animals are known for their reliability, they are also associated with high costs and time requirements. Consequently, there is an urgent demand for alternative, cost-efficient, and rapid in vitro testing methods. This study aims to address the challenges related to automating and streamlining the screening of early developmental toxicity of chemicals by introducing a mouse embryoid body test (EBT) model in a 384-ultra low attachment well format. Embryoid bodies (EBs) generated in this format were characterized by a spontaneous differentiation trajectory into cardiac mesoderm by as analyzed by RNA-seq. Assessing prediction accuracy using reference compounds suggested in the ICH S5(R3) guideline and prior studies resulted in the establishment of the acceptance criteria and applicability domain of the EBT model. The results indicated an 84.38% accuracy in predicting the developmental toxicity of 23 positive and 9 negative reference compounds, with an optimized cutoff threshold of 750 µM. Overall, the developed EBT model presents a promising approach for more rapid, high-throughput chemical screening, thereby facilitating well-informed decision-making in environmental management and safety assessments.

Npac Regulates Pre-mRNA Splicing in Mouse Embryonic Stem Cells.

As a reader of tri-methylated lysine 36 on histone H3 (H3K36me3), Npac has been shown to have a significant role in gene transcription elongation. However, its potential implication in RNA splicing remains unknown. Here, we characterized the phenotypes of Npac knockout in mES cells. We discovered that loss of Npac disrupts pluripotency and identity in mESCs. We also found that Npac is associated with many cellular activities, including cell proliferation, differentiation, and transcription regulation. Notably, we uncovered that Npac is associated with RNA splicing machinery. Furthermore, we found that Npac regulates alternative splicing through its interaction with the splicing factors, including Srsf1. Our research thus highlights the important role of Npac in maintaining ESC identity through the regulation of pre-mRNA splicing.

Integrity of the short arm of the nuclear pore Y-complex is required for mouse embryonic stem cell growth and differentiation.

Many cellular processes, ranging from cell division to differentiation, are controlled by nuclear pore complexes (NPCs). However, studying the contributions of individual NPC subunits to these processes in vertebrates has long been impeded by their complexity and the lack of efficient genetic tools. Here, we use genome editing in mouse embryonic stem cells (mESCs) to characterize the role of NPC structural components, focusing on the short arm of the Y-complex that comprises Nup85, Seh1 and Nup43. We show that Seh1 and Nup43, although dispensable in pluripotent mESCs, are required for their normal cell growth rates, their viability upon differentiation and for the maintenance of proper NPC density. mESCs with an N-terminally truncated Nup85 mutation (in which interaction with Seh1 is greatly impaired) feature a similar reduction of NPC density. However, their proliferation and differentiation are unaltered, indicating that it is the integrity of the Y-complex, rather than the number of NPCs, that is critical to ensure these processes.

Paxillin interactome identified by SILAC and label-free approaches coupled to TurboID sheds light on the compositions of focal adhesions in mouse embryonic stem cells.

Self-renewal and differentiation of mouse embryonic stem cells (mESCs) are greatly affected by the extracellular matrix (ECM) environment; the composition and stiffness of which are sensed by the cells via integrin-associated focal adhesions (FAs) which link the cells to the ECM. Although FAs have been studied extensively in differentiated cells, their composition and function in mESCs are not as well elucidated. To gain more detailed knowledge of the molecular compositions of FAs in mESCs, we adopted the proximity-dependent biotinylation (BioID) proteomics approach. Paxillin, a known FA protein (FAP), is fused to the promiscuous biotin ligase TurboID as bait. We employed both SILAC- and label-free (LF)-based quantitative proteomics to strengthen as well as complement individual approach. The mass spectrometry data derived from SILAC and LF identified 38 and 443 proteins, respectively, with 35 overlapping candidates. Fifteen of these shared proteins are known FAPs based on literature-curated adhesome and 7 others are among the reported "meta-adhesome", suggesting the components of FAs are largely conserved between mESCs and differentiated cells. Furthermore, the LF data set contained an additional 18 literature-curated FAPs. Notably, the overlapped proteomics data failed to detect LIM-domain proteins such as zyxin family proteins, which suggests that FAs in mESCs are less mature than differentiated cells. Using the LF approach, we are able to identify PDLIM7, a LIM-domain protein, as a FAP in mESCs. This study illustrates the effectiveness of TurboID in mESCs. Importantly, we found that application of both SILAC and LF methods in combination allowed us to analyze the TurboID proteomics data in an unbiased, stringent and yet comprehensive manner.

Linc1548 Promotes the Transition of Epiblast Stem Cells Into Neural Progenitors by Engaging OCT6 and SOX2.

The transition of embryonic stem cells from the epiblast stem cells (EpiSCs) to neural progenitor cells (NPCs), called the neural induction process, is crucial for cell fate determination of neural differentiation. However, the mechanism of this transition is unclear. Here, we identified a long non-coding RNA (linc1548) as a critical regulator of neural differentiation of mouse embryonic stem cells (mESCs). Knockout of linc1548 did not affect the conversion of mESCs to EpiSCs, but delayed the transition from EpiSCs to NPCs. Moreover, linc1548 interacts with the transcription factors OCT6 and SOX2 forming an RNA-protein complex to regulate the transition from EpiSCs to NPCs. Finally, we showed that Zfp521 is an important target gene of this RNA-protein complex regulating neural differentiation. Our findings prove how the intrinsic transcription complex is mediated by a lncRNA linc1548 and can better understand the intrinsic mechanism of neural fate determination.

Sall4 Guides p53-Mediated Enhancer Interference upon DNA Damage in Mouse Embryonic Stem Cells.

p53 plays a pivotal role in maintaining the genomic stability of mouse embryonic stem cells (mESCs) through transcriptionally activating and repressing target genes. However, how p53 recognizes its repressed targets remains largely unknown. Herein, we demonstrate that Sall4 negatively regulates DNA damage induced apoptosis (DIA) of mESCs through mediating p53 recruitment to enhancers of ESC-associated genes repressed by p53 from promoters of p53-activated genes. Upon DNA damage, Sall4 is transcriptionally repressed by p53 and plays an anti-apoptotic role without altering p53 activation. Moreover, Sall4 is identified as a novel p53-interacting partner. Consistently, Sall4 exerts its anti-apoptotic function in a p53-dependent manner. Intriguingly, Sall4 depletion not only promotes the transcriptional activation of several p53-regulated pro-apoptotic genes but also compromises p53-mediated repression of ESC master transcription factors in response to DNA damage. Mechanistically, Sall4 balances p53-binding affinity between p53-activated and -repressed genes through tethering p53 to ESC enhancers. In light of our study, Sall4 may contribute to tumorigenesis by antagonizing p53-mediated apoptosis.

Hypoxia is fine-tuned by Hif-1α and regulates mesendoderm differentiation through the Wnt/ß-Catenin pathway.

BACKGROUND: Hypoxia naturally happens in embryogenesis and thus serves as an important environmental factor affecting embryo development. Hif-1α, an essential hypoxia response factor, was mostly considered to mediate or synergistically regulate the effect of hypoxia on stem cells. However, the function and relationship of hypoxia and Hif-1α in regulating mesendoderm differentiation remains controversial. RESULTS: We here discovered that hypoxia dramatically suppressed the mesendoderm differentiation and promoted the ectoderm differentiation of mouse embryonic stem cells (mESCs). However, hypoxia treatment after mesendoderm was established promoted the downstream differentiation of mesendoderm-derived lineages. These effects of hypoxia were mediated by the repression of the Wnt/ß-Catenin pathway and the Wnt/ß-Catenin pathway was at least partially regulated by the Akt/Gsk3ß axis. Blocking the Wnt/ß-Catenin pathway under normoxia using IWP2 mimicked the effects of hypoxia while activating the Wnt/ß-Catenin pathway with CHIR99021 fully rescued the mesendoderm differentiation suppression caused by hypoxia. Unexpectedly, Hif-1α overexpression, in contrast to hypoxia, promoted mesendoderm differentiation and suppressed ectoderm differentiation. Knockdown of Hif-1α under normoxia and hypoxia both inhibited the mesendoderm differentiation. Moreover, hypoxia even suppressed the mesendoderm differentiation of Hif-1α knockdown mESCs, further implying that the effects of hypoxia on the mesendoderm differentiation were Hif-1α independent. Consistently, the Wnt/ß-Catenin pathway was enhanced by Hif-1α overexpression and inhibited by Hif-1α knockdown. As shown by RNA-seq, unlike hypoxia, the effect of Hif-1α was relatively mild and selectively regulated part of hypoxia response genes, which fine-tuned the effect of hypoxia on mESC differentiation. CONCLUSIONS: This study revealed that hypoxia is fine-tuned by Hif-1α and regulates the mesendoderm and ectoderm differentiation by manipulating the Wnt/ß-Catenin pathway, which contributed to the understanding of hypoxia-mediated regulation of development.

Gata6 potently initiates reprograming of pluripotent and differentiated cells to extraembryonic endoderm stem cells.

Transcription factor-mediated reprograming is a powerful method to study cell fate changes. In this study, we demonstrate that the transcription factor Gata6 can initiate reprograming of multiple cell types to induced extraembryonic endoderm stem (iXEN) cells. Intriguingly, Gata6 is sufficient to drive iXEN cells from mouse pluripotent cells and differentiated neural cells. Furthermore, GATA6 induction in human embryonic stem (hES) cells also down-regulates pluripotency gene expression and up-regulates extraembryonic endoderm (ExEn) genes, revealing a conserved function in mediating this cell fate switch. Profiling transcriptional changes following Gata6 induction in mES cells reveals step-wise pluripotency factor disengagement, with initial repression of Nanog and Esrrb, then Sox2, and finally Oct4, alongside step-wise activation of ExEn genes. Chromatin immunoprecipitation and subsequent high-throughput sequencing analysis shows Gata6 enrichment near pluripotency and endoderm genes, suggesting that Gata6 functions as both a direct repressor and activator. Together, this demonstrates that Gata6 is a versatile and potent reprograming factor that can act alone to drive a cell fate switch from diverse cell types.

Expression of Pfkfb isoenzymes during in vitro differentiation of mouse embryonic stem cells into insulin-producing cells.

Background/aim: Type 1 diabetes mellitus (T1DM) is caused by the autoimmune-mediated destruction of insulin-producing cells (IPCs) and still has no effective cure. Better understanding of the molecular mechanisms involved in the differentiation of embryonic stem cells (ESCs) into IPCs may help us improve the therapeutic strategies for treating T1DM. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (Pfkfb1-4) are key regulators of glucose metabolism. Although Pfkfb3 has been shown to be required for the growth of early differentiated mouse ESCs (mESCs), more studies are needed to further assess the roles of Pfkfb isoenzymes in embryonic development and differentiation, particularly into specific cell types. In this study, we aimed to elucidate the changes in the expression of Pfkfb isoenzymes on the differentiation of mESCs into IPCs. Materials and methods: A 3-step protocol was used to differentiate R1 and J1 mESCs into IPCs. The changes in the gene expression of MafA, MafB, Ins2, and Nkx6.1 (IPC specific markers) and Pfkfb1-4 were analyzed using real-time quantitative polymerase chain reaction (qPCR). Insulin expression and secretion were determined by immunofluorescence (IF) staining and the enzyme linked immunosorbent assay (ELISA), respectively. Results: Upon differentiation, the IPC specific markers in differentiated cells were upregulated. Continued differentiation was confirmed by the development of insulin-positive islet-like clusters that secreted insulin in response to glucose uptake. Expressions of the Pfkfb2 and Pfkfb3 isoenzymes were markedly increased in various stages of differentiation. Conclusion: These findings suggest that Pfkfb2 and Pfkfb3 may impact the differentiation of mESCs into IPCs and the regulation of the insulin response to glucose levels. This study also lays a foundation for researchers to further probe the roles of Pfkfb isoenzymes on the differentiation of mESCs into IPCs and may open new avenues for regenerative medicine.

Deciphering the role of RNA structure in translation efficiency.

BACKGROUND: RNA secondary structure has broad impact on the fate of RNA metabolism. The reduced stability of secondary structures near the translation initiation site/start codon of the coding region promotes the efficiency of translation in both prokaryotic and eukaryotic species. However, the inaccuracy of in silico folding and the focus on the coding region limit our understanding of the global relationship between the whole mRNA structure and translation efficiency. Leveraging high-throughput RNA structure probing data in the transcriptome, we aim to systematically investigate the role of RNA structure in regulating translation efficiency. RESULTS: Here, we analyze the influences of hundreds of sequence and structural features on translation efficiency in the mouse embryonic stem cells (mESCs) and zebrafish developmental stages. Our findings reveal that overall in vivo RNA structure has a higher relative importance in predicting translation efficiency than in vitro RNA structure in both mESCs and zebrafish. Also, RNA structures in 3' untranslated region (UTR) have much stronger influence on translation efficiency compared to those in coding regions or 5' UTR. Furthermore, strong alternation between in vitro and in vivo structures in 3' UTR are detected in highly translated mRNAs in mESCs but not zebrafish. Instead, moderate alteration between in vitro and in vivo RNA structures in the 5' UTR and proximal coding regions are detected in highly translated mRNAs in zebrafish. CONCLUSIONS: Our results suggest the openness of the 3' UTR promotes the translation efficiency in both mice and zebrafish, with the in vivo structure in 3' UTR more important in mice than in zebrafish. This reveals a novel role of RNA secondary structure on translational regulation.

GPR17 is an essential regulator for the temporal adaptation of sonic hedgehog signalling in neural tube development.

Dorsal-ventral pattern formation of the neural tube is regulated by temporal and spatial activities of extracellular signalling molecules. Sonic hedgehog (Shh) assigns ventral neural subtypes via activation of the Gli transcription factors. Shh activity in the neural progenitor cells changes dynamically during differentiation, but the mechanisms regulating this dynamicity are not fully understood. Here, we show that temporal change of intracellular cAMP levels confers the temporal Shh signal, and the purinergic G-protein-coupled receptor GPR17 plays an essential role in this regulation. GPR17 is highly expressed in the ventral progenitor regions of the neural tube and acts as a negative regulator of the Shh signal in chick embryos. Although the activation of the GPR17-related signal inhibits ventral identity, perturbation of Gpr17 expression leads to aberrant expansion of ventral neural domains. Notably, perturbation of Gpr17 expression partially inhibits the negative feedback of Gli activity. Moreover, GPR17 increases cAMP activity, suggesting that it exerts its function by inhibiting the processing of Gli3 protein. GPR17 also negatively regulates Shh signalling in neural cells differentiated from mouse embryonic stem cells, suggesting that GPR17 function is conserved among different organisms. Our results demonstrate that GPR17 is a novel negative regulator of Shh signalling in a wide range of cellular contexts.

Fluid shear stress promotes embryonic stem cell pluripotency via interplay between ß-catenin and vinculin in bioreactor culture.

The expansion of pluripotent stem cells (PSCs) as aggregates in stirred suspension bioreactors is garnering attention as an alternative to adherent culture. However, the hydrodynamic environment in the bioreactor can modulate PSC behavior, pluripotency and differentiation potential in ways that need to be well understood. In this study, we investigated how murine embryonic stem cells (mESCs) sense fluid shear stress and modulate a noncanonical Wnt signaling response to promote pluripotency. mESCs showed higher expression of pluripotency marker genes, Oct4, Sox2, and Nanog in the absence of leukemia inhibitory factor (LIF) in stirred suspension bioreactors compared to adherent culture, a phenomenon we have termed mechanopluripotency. In bioreactor culture, fluid shear promoted the nuclear translocation of the less well-known pluripotency regulator ß-catenin and concomitant increase of c-Myc expression, an upstream regulator of Oct4, Sox2, and Nanog. We also observed similar ß-catenin nuclear translocation in LIF-free mESCs cultured on E-cadherin substrate under defined fluid shear stress conditions in flow chamber plates. mESCs showed lower shear-induced expression of pluripotency marker genes when ß-catenin was inhibited, suggesting that ß-catenin signaling is crucial to mESC mechanopluripotency. Key to this process is vinculin, which is known to rearrange and associate more strongly with adherens junctions in response to fluid shear. When the vinculin gene is disrupted, we observe that nuclear ß-catenin translocation and mechanopluripotency are abrogated. Our results indicate that mechanotransduction through the adherens junction complex is important for mESC pluripotency maintenance.