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The discovery of embryonic stem cell (ESC) mediating tumoricidal activity revealed the intimate relationship between ESCs and tumor cells, but the functional role of ESCs in tumor progression is poorly understood. To further investigate tumor cell and ESC interactions, we co-cultured mouse ESCs with mouse pancreatic cancer Pan02 cells or mouse melanoma B16-F10 cells in Transwell, and found that tumor cell invasion was significantly inhibited by ESCs. Application of ESCs to tumor-bearing mice resulted in significant inhibition of tumor metastasis in vivo. RNA-Seq analyses of tumor cell and ESC co-cultures identified TP53 and related signalling as major pathways involved in ESC-mediated inhibition of tumor cell invasion and metastasis, which indicated the potential clinical application of ESCs to treat cancer. [ABSTRACT FROM AUTHOR]

The mammalian p57 protein is a member of the CIP/KIP family of cyclin‐dependent kinase inhibitors and plays an essential role in the development of multiple tissues during embryogenesis as well as in the maintenance of tissue stem cells in adults. Although several transcription factors have been implicated in regulating the p57 gene, cis‐elements such as enhancers that regulate its expression have remained ill‐defined. Here we identify a candidate enhancer for the mouse p57 gene (Cdkn1c) within an intron of the Kcnq1 locus by 4C‐seq analysis in mouse embryonic stem cells (mESCs). Deletion of this putative enhancer region with the CRISPR‐Cas9 system or its suppression by CRISPR interference resulted in a marked attenuation of Cdkn1c expression in differentiating mESCs. Our results thus suggest that this region may serve as an enhancer for the p57 gene during early mouse embryogenesis. [ABSTRACT FROM AUTHOR]

Stem cell‐derived cardiac spheroids are promising models for cardiac research and drug testing. However, generating contracting cardiac spheroids remains challenging because of the laborious experimental procedure. Here, we present a microfluidic hanging‐heart chip (HH‐chip) that uses a microchannel and flow‐driven system to facilitate cell loading and culture medium replacement operations to reduce the laborious manual handling involved in the generation of a large quantity of cardiac spheroids. The effectiveness of the HH‐chip was demonstrated by simultaneously forming 50 mouse embryonic stem cell‐derived embryonic bodies, which sequentially differentiated into 90% beating cardiac spheroids within 15 days of culture on the chip. A comparison of our HH‐chip method with traditional hanging‐drop and low‐attachment plate methods revealed that the HH‐chip could generate higher contracting proportions of cardiac spheroids with higher expression of cardiac markers. Additionally, we verified that the contraction frequencies of the cardiac spheroids generated from the HH‐chip were sensitive to cardiotoxic drugs. Overall, our results suggest that the microfluidic hanging drop chip‐based approach is a high‐throughput and highly efficient method for generating contracting mouse embryonic stem cell‐derived cardiac spheroids for cardiac toxicity and drug testing applications. [ABSTRACT FROM AUTHOR]

Abstract The discovery of embryonic stem cell (ESC) mediating tumoricidal activity revealed the intimate relationship between ESCs and tumor cells, but the functional role of ESCs in tumor progression is poorly understood. To further investigate tumor cell and ESC interactions, we co-cultured mouse ESCs with mouse pancreatic cancer Pan02 cells or mouse melanoma B16-F10 cells in Transwell, and found that tumor cell invasion was significantly inhibited by ESCs. Application of ESCs to tumor-bearing mice resulted in significant inhibition of tumor metastasis in vivo. RNA-Seq analyses of tumor cell and ESC co-cultures identified TP53 and related signalling as major pathways involved in ESC-mediated inhibition of tumor cell invasion and metastasis, which indicated the potential clinical application of ESCs to treat cancer.

Producing chimaeras constitutes the most reliable method of verifying the pluripotency of newly established cells. Moreover, forming chimaeras by injecting genetically modified embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) into the embryo is part of the procedure for generating transgenic mice, which are used for understanding gene function. Conventional methods for generating transgenic mice, including the breeding of chimaeras and tetraploid complementation, are time-consuming and cost-inefficient, with significant limitations that hinder their effectiveness and widespread applications. In the present study, we modified the traditional method of chimaera generation to significantly speed up this process by generating mice exclusively derived from ESCs. This study aimed to assess whether fully ESC-derived mice could be obtained by modulating fibroblast growth factor 4 (FGF4) levels in the culture medium and changing the direction of cell differentiation in the chimaeric embryo. We found that exogenous FGF4 directs all host blastomeres to the primitive endoderm fate, but does not affect the localisation of ESCs in the epiblast of the chimaeric embryos. Consequently, all FGF4-treated chimaeric embryos contained an epiblast composed exclusively of ESCs, and following transfer into recipient mice, these embryos developed into fully ESC-derived newborns. Collectively, this simple approach could accelerate the generation of ESC-derived animals and thus optimise ESC-mediated transgenesis and the verification of cell pluripotency. Compared to traditional methods, it could speed up functional studies by several weeks and significantly reduce costs related to maintaining and breeding chimaeras. Moreover, since the effect of stimulating the FGF signalling pathway is universal across different animal species, our approach can be applied not only to rodents but also to other animals, offering its utility beyond laboratory settings. • FGF4-treated ESC chimaeric embryos contain an epiblast composed exclusively of ESCs. • After transfer into recipient mice, embryos develop into fully ESC-derived newborns. • Supplementing culture medium with FGF4 can accelerate the generation of ESC-derived mice. • FGF4 supplementation can optimise the procedure of verifying cell pluripotency. [ABSTRACT FROM AUTHOR]

As the most prevalent and reversible internal epigenetic modification in eukaryotic mRNAs, N6-methyladenosine (m6A) post-transcriptionally regulates the processing and metabolism of mRNAs involved in diverse biological processes. m6A modification is regulated by m6A writers, erasers, and readers. Emerging evidence suggests that m6A modification plays essential roles in modulating the cell-fate transition of embryonic stem cells. Mechanistic investigation of embryonic stem cell maintenance and differentiation is critical for understanding early embryonic development, which is also the premise for the application of embryonic stem cells in regenerative medicine. This review highlights the current knowledge of m6A modification and its essential regulatory contribution to the cell fate transition of mouse and human embryonic stem cells.

Tissue fibrosis following tendon injury is a major clinical problem due to the increased risk of re-injury and limited treatment options; however, its mechanism remains unclear. Evidence suggests that insufficient resolution of inflammation contributes to fibrotic healing by disrupting tenocyte activity, with the NF-κB pathway being identified as a potential mediator. Equine embryonic stem cell (ESC) derived tenocytes may offer a potential cell-based therapy to improve tendon regeneration, but how they respond to an inflammatory environment is largely unknown. Our findings reveal for the first time that, unlike adult tenocytes, ESC-tenocytes are unaffected by IFN-γ, TNFα, and IL-1β stimulation; producing minimal changes to tendon-associated gene expression and generating 3-D collagen gel constructs indistinguishable from unstimulated controls. Inflammatory pathway analysis found these inflammatory cytokines failed to activate NF-κB in the ESC-tenocytes. However, NF-κB could be activated to induce changes in gene expression following stimulation with NF-κB pharmaceutical activators. Transcriptomic analysis revealed differences between cytokine and NF-κB signalling components between adult and ESC-tenocytes, which may contribute to the mechanism by which ESC-tenocytes escape inflammatory stimuli. Further investigation of these molecular mechanisms will help guide novel therapies to reduce fibrosis and encourage superior tendon healing. [ABSTRACT FROM AUTHOR]

Cell therapy is one of the promising approaches used against type 1 diabetes. Efficient generation of human embryonic stem cell (hESC)-derived pancreatic progenitors (PPs) is of great importance. Since signaling pathways underlying human pancreas development are not yet fully understood, various differentiation protocols are conducted, each considering variable duration, timing, and concentrations of growth factors and small molecules. Therefore, we compared two PP differentiation protocols in static suspension culture. We tested modified protocols developed by Pagliuca et al. (protocol 1) and Royan researchers (protocol 2) until early PP stage. The morphological changes of hESC aggregates during differentiation, and also gene and protein expression after differentiation, were evaluated. Different morphological structures were formed in each protocol. Quantitative gene expression analysis, flow cytometry, and immunostaining revealed a high level of PDX1 expression on day 13 of Royan's differentiation protocol compared to protocol 1. Our data showed that using protocol 2, cells were further differentiated until day 16, showing higher efficiency of early PPs. Moreover, protocol 2 is able to produce hESCs-PPs in a static suspension culture. Since protocol 2 is inexpensive in terms of media, growth factors, and chemicals, it can be used for massive production of PPs using static and dynamic suspension cultures. [ABSTRACT FROM AUTHOR]

Abstract Embryonic stem cell (ESC)-derived epitopes can act as therapeutic tumor vaccines against different types of tumors Jin (Adv Healthc Mater 2023). However, these epitopes have poor immunogenicity and stimulate insufficient CD8+ T cell responses, which motivated us to develop a new method to deliver and enhance their effectiveness. Bacterial outer membrane vesicles (OMVs) can serve as immunoadjuvants and act as a delivery vector for tumor antigens. In the current study, we engineered a new OMV platform for the co-delivery of ESC-derived tumor antigens and immune checkpoint inhibitors (PD-L1 antibody). An engineered Staphylococcal Protein A (SpA) was created to non-specifically bind to anti-PD-L1 antibody. SpyCatcher (SpC) and SpA were fused into the cell outer membrane protein OmpA to capture SpyTag-attached peptides and PD-L1 antibody, respectively. The modified OMV was able to efficiently conjugate with ESC-derived TAAs and PD-L1 antibody (SpC-OMVs + SpT-peptides + anti-PD-L1), increasing the residence time of TAAs in the body. The results showed that the combination therapy of ESC-based TAAs and PD-L1 antibody delivered by OMV had significant inhibitory effects in mouse tumor model. Specifically, it was effective in reducing tumor growth by enhancing IFN-γ-CD8+ T cell responses and increasing the number of CD8+ memory cells and antigen-specific T cells. Overall, the new OMV delivery system is a versatile platform that can enhance the immune responses of ESC-based TAA cancer vaccines. Graphical Abstract

Abstract In recent years, extracellular vesicles (EVs) have attracted significant attention as carriers in intercellular communication. The vast array of information contained within EVs is critical for various cellular activities, such as proliferation and differentiation of multiple cell types. Moreover, EVs are being employed in disease diagnostics, implicated in disease etiology, and have shown promise in tissue repair. Recently, a phenomenon has been discovered in which cellular phenotypes, including the progression of differentiation, are synchronized among cells via EVs. This synchronization could be prevalent in widespread different situations in embryogenesis and tissue organization and maintenance. Given the increasing research on multi-cellular tissues and organoids, the role of EV-mediated intercellular communication has become increasingly crucial. This review begins with fundamental knowledge of EVs and then discusses recent findings, various modes of information transfer via EVs, and synchronization of cellular phenotypes.

Abstract Background Humans’ nervous system has a limited ability to repair nerve cells, which poses substantial challenges in treating injuries and diseases. Stem cells are identified by the potential to renew their selves and develop into several cell types, making them ideal candidates for cell replacement in injured neurons. Neuronal differentiation of embryonic stem cells in modern medicine is significant. Nanomaterials have distinct advantages in directing stem cell function and tissue regeneration in this field. We attempted in this systematic review to collect data, analyze them, and report results on the effect of nanomaterials on neuronal differentiation of embryonic stem cells. Methods International databases such as PubMed, Scopus, ISI Web of Science, and EMBASE were searched for available articles on the effect of nanomaterials on neuronal differentiation of embryonic stem cells (up to OCTOBER 2023). After that, screening (by title, abstract, and full text), selection, and data extraction were performed. Also, quality assessment was conducted based on the STROBE checklist. Results In total, 1507 articles were identified and assessed, and then only 29 articles were found eligible to be included. Nine studies used 0D nanomaterials, ten used 1D nanomaterials, two reported 2D nanomaterials, and eight demonstrated the application of 3D nanomaterials. The main biomaterial in studies was polymer-based composites. Three studies reported the negative effect of nanomaterials on neural differentiation. Conclusion Neural differentiation is crucial in neurological regenerative medicine. Nanomaterials with different characteristics, particularly those cellular regulating activities and stem cell fate, have much potential in neural tissue engineering. These findings indicate a new understanding of potential applications of physicochemical cues in nerve tissue engineering. Graphical Abstract

Cardiac regenerative therapy using human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is expected to become an alternative to heart transplantation for severe heart failure. It is now possible to produce large numbers of human pluripotent stem cells (hPSCs) and eliminate non-cardiomyocytes, including residual undifferentiated hPSCs, which can cause teratoma formation after transplantation. There are two main strategies for transplanting hPSC-CMs: injection of hPSC-CMs into the myocardium from the epicardial side, and implantation of hPSC-CM patches or engineered heart tissues onto the epicardium. Transplantation of hPSC-CMs into the myocardium of large animals in a myocardial infarction model improved cardiac function. The engrafted hPSC-CMs matured, and microvessels derived from the host entered the graft abundantly. Furthermore, as less invasive methods using catheters, injection into the coronary artery and injection into the myocardium from the endocardium side have recently been investigated. Since transplantation of hPSC-CMs alone has a low engraftment rate, various methods such as transplantation with the extracellular matrix or non-cardiomyocytes and aggregation of hPSC-CMs have been developed. Post-transplant arrhythmias, imaging of engrafted hPSC-CMs, and immune rejection are the remaining major issues, and research is being conducted to address them. The clinical application of cardiac regenerative therapy using hPSC-CMs has just begun and is expected to spread widely if its safety and efficacy are proven in the near future. • hPSC-CM transplantation improves cardiac function of infarcted animal hearts. • Several administration methods using catheters or injection devices were developed. • Post-transplant arrhythmia is one of the critical hurdles in hPSC-CM transplantation therapy. • Cardiac regenerative therapy using hPSC-CMs has recently been applied in humans. [ABSTRACT FROM AUTHOR]

Introduction: We have previously observed phenotypic and developmental changes upon the ectopic expression of the RUNX3 or the ZBTB46 transcription factors in mouse embryonic stem cell (ESC) derived progenitors. In this study, we evaluated the gene expression profiles of the RUNX3- and the ZBTB46-instructed murine ESCs with RNA-seq testing two next-generation sequencing technologies. Methods: We compared the DNA nanoball-based DNBSEQ G400 sequencer (MGI) with the bridge-PCR-based NextSeq 500 instrument (Illumina) for RNA sequencing. Moreover, we also compared two types of MGI sequencing reagents (Standard versus Hot-massive parallel sequencing (MPS)) with the DNBSEQ G400. Results: We observed that both sequencing platforms showed comparable levels of quality, sequencing uniformity, and gene expression profiles. For example, highly overlapping RUNX3- and ZBTB46-regulated gene lists were obtained from both sequencing datasets. Moreover, we observed that the Standard and the Hot-MPS-derived RUNX3- and ZBTB46-regulated gene lists were also considerably overlapped. This transcriptome analysis also helped us to identify differently expressed genes in the presence of the transgenic RUNX3 or ZBTB46. For example, we found that Gzmb, Gzmd, Gzme, Gdf6, and Ccr7 genes were robustly upregulated upon the forced expression of Runx3; on the other hand, Gpx2, Tdpoz4, and Arg2 were induced alongside the ectopic expression of Zbtb46. Discussion: Similar gene expression profile and greatly overlapping RUNX3- and ZBTB46-regulated gene sets were detected with both DNA sequencing platforms. Our analyses demonstrate that both sequencing technologies are suitable for transcriptome profiling and target gene selection. These findings suggest that DNBSEQ G400 represents a cost-effective alternative sequencing platform for gene expression monitoring. Moreover, this analysis provides a resource for exploration of the RUNX3- and ZBTB46-dependent gene regulatory networks. [ABSTRACT FROM AUTHOR]

Embryonic stem cell (ESC)-derived epitopes can act as therapeutic tumor vaccines against different types of tumors Jin (Adv Healthc Mater 2023). However, these epitopes have poor immunogenicity and stimulate insufficient CD8+ T cell responses, which motivated us to develop a new method to deliver and enhance their effectiveness. Bacterial outer membrane vesicles (OMVs) can serve as immunoadjuvants and act as a delivery vector for tumor antigens. In the current study, we engineered a new OMV platform for the co-delivery of ESC-derived tumor antigens and immune checkpoint inhibitors (PD-L1 antibody). An engineered Staphylococcal Protein A (SpA) was created to non-specifically bind to anti-PD-L1 antibody. SpyCatcher (SpC) and SpA were fused into the cell outer membrane protein OmpA to capture SpyTag-attached peptides and PD-L1 antibody, respectively. The modified OMV was able to efficiently conjugate with ESC-derived TAAs and PD-L1 antibody (SpC-OMVs + SpT-peptides + anti-PD-L1), increasing the residence time of TAAs in the body. The results showed that the combination therapy of ESC-based TAAs and PD-L1 antibody delivered by OMV had significant inhibitory effects in mouse tumor model. Specifically, it was effective in reducing tumor growth by enhancing IFN-γ-CD8+ T cell responses and increasing the number of CD8+ memory cells and antigen-specific T cells. Overall, the new OMV delivery system is a versatile platform that can enhance the immune responses of ESC-based TAA cancer vaccines. [ABSTRACT FROM AUTHOR]

Polycystic ovary syndrome (PCOS) is the most prevalent endocrine condition among women with pleiotropic sequelae possessing reproductive, metabolic, and psychological characteristics. Although the exact origin of PCOS is elusive, it is known to be a complex multigenic disorder with a genetic, epigenetic, and environmental background. However, the pathogenesis of PCOS, and the role of genetic variants in increasing the risk of the condition, are still unknown due to the lack of an appropriate study model. Since the debut of induced pluripotent stem cell (iPSC) technology, the ability of reprogrammed somatic cells to self-renew and their potential for multidirectional differentiation have made them excellent tools to study different disease mechanisms. Recently, researchers have succeeded in establishing human in vitro PCOS disease models utilizing iPSC lines from heterogeneous PCOS patient groups (iPSCPCOS). The current review sets out to summarize, for the first time, our current knowledge of the implications and challenges of iPSC technology in comprehending PCOS pathogenesis and tissue-specific disease mechanisms. Additionally, we suggest that the analysis of polygenic risk prediction based on genome-wide association studies (GWAS) could, theoretically, be utilized when creating iPSC lines as an additional research tool to identify women who are genetically susceptible to PCOS. Taken together, iPSCPCOS may provide a new paradigm for the exploration of PCOS tissue-specific disease mechanisms. [ABSTRACT FROM AUTHOR]

Conventional cell biology studies focus on cellular responses to chemical signals, but cells also react to mechanical cues like density, size, and substrate rigidity, activating specific gene expression. Embryo development leads to the formation of a gastrula, establishing body structure and germ layers (endoderm, ectoderm, mesoderm) via diverse mechanisms. In humans, gastrulation begins with the Primitive Streak (PS) and T gene expression, guiding epiblast cell migration. Self-regulation occurs in gastruloid models, derived from human embryonic stem cells, capable of differentiation. Mediators like YAP/TAZ and PIEZO1 link density to cellular responses, with ETV4 serving as a link between mechanical environment and gene expression. This research employed a systematic literature review to synthesize relevant studies. Inspired by stem cell advancements, particularly ETV4's role, searches on PubMed yielded three articles meeting inclusion criteria. ES cells maintain undifferentiated states via ETV4 and ETV5. Rapid cell growth deactivates ETV4, prompting differentiation, influenced by mechanical cues. ETV4, ETV5, and SPRY4 regulate the FGF/ERK pathway, modulating sensitivity. High density initiates neuroectodermal cell formation, impacting integrin-actomyosin and FGFR pathways, via ETV4. Fluctuations in density dictate lineage fate, with ETV4 as a key sensor, linking density shifts to lineage determination via the ERK pathway. [ABSTRACT FROM AUTHOR]

Background: Humans' nervous system has a limited ability to repair nerve cells, which poses substantial challenges in treating injuries and diseases. Stem cells are identified by the potential to renew their selves and develop into several cell types, making them ideal candidates for cell replacement in injured neurons. Neuronal differentiation of embryonic stem cells in modern medicine is significant. Nanomaterials have distinct advantages in directing stem cell function and tissue regeneration in this field. We attempted in this systematic review to collect data, analyze them, and report results on the effect of nanomaterials on neuronal differentiation of embryonic stem cells. Methods: International databases such as PubMed, Scopus, ISI Web of Science, and EMBASE were searched for available articles on the effect of nanomaterials on neuronal differentiation of embryonic stem cells (up to OCTOBER 2023). After that, screening (by title, abstract, and full text), selection, and data extraction were performed. Also, quality assessment was conducted based on the STROBE checklist. Results: In total, 1507 articles were identified and assessed, and then only 29 articles were found eligible to be included. Nine studies used 0D nanomaterials, ten used 1D nanomaterials, two reported 2D nanomaterials, and eight demonstrated the application of 3D nanomaterials. The main biomaterial in studies was polymer-based composites. Three studies reported the negative effect of nanomaterials on neural differentiation. Conclusion: Neural differentiation is crucial in neurological regenerative medicine. Nanomaterials with different characteristics, particularly those cellular regulating activities and stem cell fate, have much potential in neural tissue engineering. These findings indicate a new understanding of potential applications of physicochemical cues in nerve tissue engineering. [ABSTRACT FROM AUTHOR]

Background: Coating a culture plate with molecules that aid in cell adhesion is a technique widely used to produce animal cell cultures. Extracellular matrix (ECM) is known for its efficiency in promoting adhesion, survival, and proliferation of adherent cells. Gelatin, a cost-effective type of ECM, is widely used in animal cell cultures including feeder-free embryonic stem (ES) cells. However, the optimal concentration of gelatin is a point of debate among researchers, with no studies having established the optimal gelatin concentration. Methods: In this study, we coated plastic plates with gelatin in a concentrationdependent manner and assessed Young’s modulus using atomic force microscopy (AFM) to investigate the microstructure of the surface of each plastic plate. The adhesion, proliferation, and differentiation of the ESCs were compared and analyzed revealing differences in surface microstructure dependent on coating concentration. Results: According to AFM analysis, there was a clear difference in the microstructure of the surface according to the presence or absence of the gelatin coating, and it was confirmed that there was no difference at a concentration of 0.5% or more. ES cell also confirmed the difference in cell adhesion, proliferation, and differentiation according to the presence or absence of gelatin coating, and also it showed no difference over the concentration of 0.5%. Conclusions: The optimum gelatin-coating for the maintenance and differentiation of ES cells is 0.5%, and the gelatin concentration-mediated microenvironment and ES cell signaling are closely correlated.

Abstract Background Prenatal infection has been implicated in the development of neuropsychiatric disorders in children. We hypothesised that exposure to lipopolysaccharide during prenatal development could induce anxiety-like behaviour and sensorineural hearing loss in offspring, as well as disrupt neural differentiation during embryonic neural development. Methods We simulated prenatal infection in FVB mice and mouse embryonic stem cell (ESC) lines, specifically 46C and E14Tg2a, through lipopolysaccharide treatment. Gene expression profiling analyses and behavioural tests were utilized to study the effects of lipopolysaccharide on the offspring and alterations in toll-like receptor (TLR) 2-positive and TLR4-positive cells during neural differentiation in the ESCs. Results Exposure to lipopolysaccharide (25 µg/kg) on gestation day 9 resulted in anxiety-like behaviour specifically in male offspring, while no effects were detected in female offspring. We also found significant increases in the expression of GFAP and CNPase, as well as higher numbers of GFAP + astrocytes and O4+ oligodendrocytes in the prefrontal cortex of male offspring. Furthermore, increased scores for genes related to oligodendrocyte and lipid metabolism, particularly ApoE, were observed in the prefrontal cortex regions. Upon exposure to lipopolysaccharide during the ESC-to-neural stem cell (NSC) transition, Tuj1, Map2, Gfap, O4, and Oligo2 mRNA levels increased in the differentiated neural cells on day 14. In vitro experiments demonstrated that lipopolysaccharide exposure induced inflammatory responses, as evidenced by increased expression of IL1b and ApoB mRNA. Conclusions Our findings suggest that prenatal infection at different stages of neural differentiation may result in distinct disturbances in neural differentiation during ESC—NSC transitions. Furthermore, early prenatal challenges with lipopolysaccharide selectively induce anxiety-like behaviour in male offspring. This behaviour may be attributed to the abnormal differentiation of astrocytes and oligodendrocytes in the brain, potentially mediated by ApoB/E signalling pathways in response to inflammatory stimuli.

Introduction: We have previously observed phenotypic and developmental changes upon the ectopic expression of the RUNX3 or the ZBTB46 transcription factors in mouse embryonic stem cell (ESC) derived progenitors. In this study, we evaluated the gene expression profiles of the RUNX3- and the ZBTB46-instructed murine ESCs with RNA-seq testing two next-generation sequencing technologies.Methods: We compared the DNA nanoball-based DNBSEQ G400 sequencer (MGI) with the bridge-PCR-based NextSeq 500 instrument (Illumina) for RNA sequencing. Moreover, we also compared two types of MGI sequencing reagents (Standard versus Hot-massive parallel sequencing (MPS)) with the DNBSEQ G400.Results: We observed that both sequencing platforms showed comparable levels of quality, sequencing uniformity, and gene expression profiles. For example, highly overlapping RUNX3- and ZBTB46-regulated gene lists were obtained from both sequencing datasets. Moreover, we observed that the Standard and the Hot-MPS-derived RUNX3- and ZBTB46-regulated gene lists were also considerably overlapped. This transcriptome analysis also helped us to identify differently expressed genes in the presence of the transgenic RUNX3 or ZBTB46. For example, we found that Gzmb, Gzmd, Gzme, Gdf6, and Ccr7 genes were robustly upregulated upon the forced expression of Runx3; on the other hand, Gpx2, Tdpoz4, and Arg2 were induced alongside the ectopic expression of Zbtb46.Discussion: Similar gene expression profile and greatly overlapping RUNX3- and ZBTB46-regulated gene sets were detected with both DNA sequencing platforms. Our analyses demonstrate that both sequencing technologies are suitable for transcriptome profiling and target gene selection. These findings suggest that DNBSEQ G400 represents a cost-effective alternative sequencing platform for gene expression monitoring. Moreover, this analysis provides a resource for exploration of the RUNX3- and ZBTB46-dependent gene regulatory networks.

Gene editing in farm animals has produced a range of genetically sterilised hosts for complementation with elite donor cells. The most promising editable target gene is NANOS2 , which allows efficient breeding of a continuous supply of sterile male hosts. Competing complementation approaches have been used to restore the missing germlines by transplanting either spermatogonial stem cells (SSCs) into the testis or embryonic stem cells (ESCs) into early embryos. Both approaches are designed to produce animals that exclusively transmit the elite male donor genotype, but not the germline-disabling mutation, to their offspring. Additional biological barriers to testis colonisation and germline restoration after SSC transplantation were recently identified in mice. Pluripotent ESCs from different livestock species show extensive proliferation, gene editing potential, and a contribution to somatic but not germline chimaeras. Animal breeding drives genetic progress mainly through the male germline. This process is slow to respond to rapidly mounting environmental pressures that threaten sustainable food security from animal protein production. New approaches promise to accelerate breeding by producing chimaeras, which comprise sterile host and fertile donor genotypes, to exclusively transmit elite male germlines. Following gene editing to generate sterile host cells, the missing germline can be restored by transplanting either: (i) spermatogonial stem cells (SSCs) into the testis; or (ii) embryonic stem cells (ESCs) into early embryos. Here we compare these alternative germline complementation strategies and their impact on agribiotechnology and species conservation. We propose a novel breeding platform that integrates embryo-based complementation with genomic selection, multiplication, and gene modification. [ABSTRACT FROM AUTHOR]

Genomes comprise a large fraction of repetitive sequences folded into constitutive heterochromatin, which protect genome integrity and cell identity. De novo formation of heterochromatin during preimplantation development is an essential step for preserving the ground-state of pluripotency and the self-renewal capacity of embryonic stem cells (ESCs). However, the molecular mechanisms responsible for the remodeling of constitutive heterochromatin are largely unknown. Here, we identify that DAXX, an H3.3 chaperone essential for the maintenance of mouse ESCs in the ground state, accumulates in pericentromeric regions independently of DNA methylation. DAXX recruits PML and SETDB1 to promote the formation of heterochromatin, forming foci that are hallmarks of ground-state ESCs. In the absence of DAXX or PML, the threedimensional (3D) architecture and physical properties of pericentric and peripheral heterochromatin are disrupted, resulting in derepression of major satellite DNA, transposable elements and genes associated with the nuclear lamina. Using epigenome editing tools, we observe that H3.3, and specifically H3.3K9 modification, directly contribute to maintaining pericentromeric chromatin conformation. Altogether, our data reveal that DAXX is crucial for the maintenance and 3D organization of the heterochromatin compartment and protects ESC viability. [ABSTRACT FROM AUTHOR]

Fetal development is one of the most sensitive windows to methylmercury (MeHg) toxicity. Laboratory and epidemiological studies have shown a dose–response relationship between fetal MeHg exposure and neuro performance in different life stages from infants to adults. In addition, MeHg exposure has been reported to be associated with disorders in endoderm-derived organs, such as morphological changes in liver cells and pancreatic cell dysfunctions. However, the mechanisms of the effects of MeHg on non-neuronal organs or systems, especially during the early development of endoderm-derived organs, remain unclear. Here we determined the effects of low concentrations of MeHg exposure during the differentiation of definitive endoderm (DE) cells from human embryonic stem cells (hESCs). hESCs were exposed to MeHg (0, 10, 100, and 200 nM) that covers the range of Hg concentrations typically found in human maternal blood during DE cell induction. Transcriptomic analysis showed that sub-lethal doses of MeHg exposure could alter global gene expression patterns during hESC to DE cell differentiation, leading to increased expression of endodermal genes/proteins and the over-promotion of endodermal fate, mainly through disrupting calcium homeostasis and generating ROS. Bioinformatic analysis results suggested that MeHg exerts its developmental toxicity mainly by disrupting ribosome biogenesis during early cell lineage differentiation. This disruption could lead to aberrant growth or dysfunctions of the developing endoderm-derived organs, and it may be the underlying mechanism for the observed congenital diseases later in life. Based on the results, we proposed an adverse outcome pathway for the effects of MeHg exposure during human embryonic stem cells to definitive endoderm differentiation. [ABSTRACT FROM AUTHOR]

Nanog and Oct4 are core transcription factors that form part of a gene regulatory network to regulate hundreds of target genes for pluripotency maintenance in mouse embryonic stem cells (ESCs). To understand their function in the pluripotency maintenance, we visualised and quantified the dynamics of single molecules of Nanog and Oct4 in a mouse ESCs during pluripotency loss. Interestingly, Nanog interacted longer with its target loci upon reduced expression or at the onset of differentiation, suggesting a feedback mechanism to maintain the pluripotent state. The expression level and interaction time of Nanog and Oct4 correlate with their fluctuation and interaction frequency, respectively, which in turn depend on the ESC differentiation status. The DNA viscoelasticity near the Oct4 target locus remained flexible during differentiation, supporting its role either in chromatin opening or a preferred binding to uncondensed chromatin regions. Based on these results, we propose a new negative feedback mechanism for pluripotency maintenance via the DNA condensation state‐dependent interplay of Nanog and Oct4. Synopsis: The transcription factors Nanog and Oct4 are core pluripotency regulators in embryonic stem cells (ESCs). Here, single‐molecule live imaging in mouse ESCs shows that interaction of Nanog and Oct4 with their target loci is regulated by the mechanical properties of DNA and suggests a new negative feedback mechanism for pluripotency maintenance. Nanog interaction with its target loci is prolonged upon reduced Nanog expression and at the onset of differentiation.DNA viscoelasticity measurements show that DNA remains flexible near the Oct4 target locus as differentiation begins.Dissociation rates of both Nanog and Oct4 correlate with the mechanical properties of DNA near their binding sites.Expression levels of Nanog and Oct4 affect the local flexibility of DNA and their DNA interaction probability. [ABSTRACT FROM AUTHOR]

Pluripotent stem cells possess the potential to differentiate into all three germ layers. However, upon removal of the stemness factors, pluripotent stem cells, such as embryonic stem cells (ESCs), exhibit EMT-like cell behavior and lose stemness signatures. This process involves the membrane translocation of the t-SNARE protein syntaxin4 (Stx4) and the expression of the intercellular adhesion molecule P-cadherin. The forced expression of either of these elements induces the emergence of such phenotypes even in the presence of stemness factors. Interestingly, extracellular Stx4, but not P-cadherin, appears to induce a significant upregulation of the gastrulation-related gene brachyury , along with a slight upregulation of the smooth muscle cell-related gene ACTA2 in ESCs. Furthermore, our findings reveal that extracellular Stx4 plays a role in preventing the elimination of CCAAT enhancer binding protein β (C/EBPβ). Notably, the forced overexpression of C/EBPβ led to the downregulation of brachyury and a significant upregulation of ACTA2 in ESCs. These observations suggest that extracellular Stx4 contributes to early mesoderm induction while simultaneously activating an element that alters the differentiation state. The fact that a single differentiation cue can elicit multiple differentiation responses may reflect the challenges associated with achieving sensitive and directed differentiation in cultured stem cells. [Display omitted] • ES cells spontaneously extrude syntaxin4, which disrupts stem cell signatures. • Extracellular syntaxin4 elicits multiple differentiation responses in ES cells. • Extracellular syntaxin4 activates P-cadherin-dependent and -independent pathways. • Extracellular syntaxin4 enables the expression of a CEBPβ gene product, LAP. • LAP protein interferes with the primary effect of extracellular syntaxin4. [ABSTRACT FROM AUTHOR]

Dax1 (Nr0b1; Dosage-sensitive sex reversal-adrenal hypoplasia congenital on the X-chromosome gene-1) is an important component of the transcription factor network that governs pluripotency in mouse embryonic stem cells (ESCs). Functional evaluation of alternative splice variants of pluripotent transcription factors has shed additional insight on the maintenance of ESC pluripotency and self-renewal. Dax1 splice variants have not been identified and characterized in mouse ESCs. We identified 18 new transcripts of Dax1 with putative protein-coding properties and compared their protein structures with known Dax1 protein (Dax1-472). The expression pattern analysis showed that the novel isoforms were cotranscribed with Dax1-472 in mouse ESCs, but they had transcriptional heterogeneity among single cells and the subcellular localization of the encoded proteins differed. Cell function experiments indicated that Dax1-404 repressed Gata6 transcription and functionally replaced Dax1-472, while Dax1-38 and Dax1-225 partially antagonized Dax1-472 transcriptional repression. This study provided a comprehensive characterization of the Dax1 splice variants in mouse ESCs and suggested complex effects of Dax1 variants in a self-renewal regulatory network. [ABSTRACT FROM AUTHOR]

This research was conducted to investigate the growth performance of black-boned chicken embryonic stem (ES) cells for the future development of cultured meat. Black-boned chicken ES cells were isolated and cultured from fertilized eggs. The treatments applied were: fetal bovine serum (FBS) (T1), commercial chicken serum (SCK) (T2), Pradu Hang Dam chicken serum (PDC) (T3), and black-boned chicken serum (BBC) (T4). Black-boned chicken ES cells were cultured at 37.0 °C in a humidified environment of 5% CO2, for 10 days. The growth of black-boned chicken ES cells concentration was measured by the absorbance at 450 nm. A haemocytometer was used to count the number of black-boned chicken ES cells. Comparing the protein content of cultured meat and chicken meat was collected for combustion and proximate analysis. All collected data were analyzed using ANOVA in a completely randomized design. T4 tended to have a higher number and growth rate than the other groups, followed by T3, T2, and T1, respectively. When counted ES cells final, T4 had a significantly higher number and growth rate than the other groups (p<0.001). Comparing the protein content, it was found that cultured meat had significantly more protein than the other groups (p<0.001), and characteristics (alignment and cross-section of muscle fibers) were not different from chicken cells. Finally, the cultivation of black-boned chicken ES cells necessitates the use of a medium containing black-boned chicken serum in the growth and development of black-boned Chicken ES cells for culture meat. [ABSTRACT FROM AUTHOR]

Objective: Proper deactivation of the pluripotency network and activation of a lineage-specific gene expression program are critical for mouse embryonic stem cell (mESC) differentiation. This is achieved by the coordinated action of transcription and chromatin factors. Our previous work identified ARID4B as a critical chromatin factor for mesoderm and endoderm differentiation. As part of a histone deacetylase complex, ARID4B plays a role in transcriptional suppression of its direct targets. Here, we investigated the mechanism of ARID4B function in mESC differentiation by focusing on genes and pathways that are upregulated in its absence. Material and Methods: We analyzed transcriptomic results of wild-type and arid4bA endoderm or mesoderm differentiated cells through integrative genomics viewer and ingenuity pathway analysis. We performed real-time quantitative polymerase chain reaction for selected genes. To understand pathway activation, we performed Western blot for candidate proteins during the time-course of differentiation. We also analyzed H3K4me3, H3K27me3 and H3K27Ac ChlP-seq results to understand changes in the chromatin environment. Results: Interferon-related genes were activated in arid4bA mESCs and endoderm or mesoderm directed cells. Consistent with this, higher phosphorylated STAT1 levels were found in arid4bA mESCs while a related phosphorylated STAT3 was unchanged. Finally, we observed a significant increase in H3K4me3 around interferon-related distal gene regulatory regions with a combination of either upregulation of H3K27Ac level or downregulation of H3K27me3 level. Conclusion: These results provide evidence that ARID4B is involved in the suppression of interferon-related genes in mESCs and during meso/ endoderm differentiation through modulation, mainly of H3K4me3. This regulation might be important for successful mESC differentiation. [ABSTRACT FROM AUTHOR]

Abstract Background Post-translational modifications of proteins are crucial to the regulation of their activity and function. As a newly discovered acylation modification, crotonylation of non-histone proteins remains largely unexplored, particularly in human embryonic stem cells (hESCs). Methods We investigated the role of crotonylation in hESC differentiation by introduce crotonate into the culture medium of GFP tagged LTR7 primed H9 cell and extended pluripotent stem cell lines. RNA-seq assay was used to determine the hESC transcriptional features. Through morphological changes, qPCR of pluripotent and germ layer-specific gene markers and flow cytometry analysis, we determined that the induced crotonylation resulted in hESC differentiating into the endodermal lineage. We performed targeted metabolomic analysis and seahorse metabolic measurement to investigate the metabolism features after crotonate induction. Then high-resolution tandem mass spectrometry (LC–MS/MS) revealed the target proteins in hESCs. In addition, the role of crotonylated glycolytic enzymes (GAPDH and ENOA) was evaluated by in vitro crotonylation and enzymatic activity assays. Finally, we used knocked-down hESCs by shRNA, wild GAPDH and GAPDH mutants to explore potential role of GAPDH crotonylation in regulating human embryonic stem cell differentiation and metabolic switch. Result We found that induced crotonylation in hESCs resulted in hESCs of different pluripotency states differentiating into the endodermal lineage. Increased protein crotonylation in hESCs was accompanied by transcriptomic shifts and decreased glycolysis. Large-scale crotonylation profiling of non-histone proteins revealed that metabolic enzymes were major targets of inducible crotonylation in hESCs. We further discovered GAPDH as a key glycolytic enzyme regulated by crotonylation during endodermal differentiation from hESCs. Conclusions Crotonylation of GAPDH decreased its enzymatic activity thereby leading to reduced glycolysis during endodermal differentiation from hESCs.

Stem cell factor (SCF), a cytokine growth factor, is expressed in various tissues of the male and female reproductive organs, including the testis, ovary, and endometrium. Its primary function involves cell survival, differentiation, and proliferation, achieved through its binding to the c-kit receptor. This study aimed to scrutinize the effects of SCF treatment during in vitro culture (IVC) on both the developmental potential and the efficiency of establishing embryonic stem cells (ESCs) from fertilized and cloned porcine embryos. The rates of cleavage and blastocyst formation exhibited no significant differences between fertilized and cloned embryos, even with the addition of SCF. However, it’s worth noting that embryos cloned with Cloud eGFP as donor cells demonstrated notably increased rates of hatched blastocysts when treated with SCF, and this increase was statistically significant (p

Background: Prenatal infection has been implicated in the development of neuropsychiatric disorders in children. We hypothesised that exposure to lipopolysaccharide during prenatal development could induce anxiety-like behaviour and sensorineural hearing loss in offspring, as well as disrupt neural differentiation during embryonic neural development. Methods: We simulated prenatal infection in FVB mice and mouse embryonic stem cell (ESC) lines, specifically 46C and E14Tg2a, through lipopolysaccharide treatment. Gene expression profiling analyses and behavioural tests were utilized to study the effects of lipopolysaccharide on the offspring and alterations in toll-like receptor (TLR) 2-positive and TLR4-positive cells during neural differentiation in the ESCs. Results: Exposure to lipopolysaccharide (25 µg/kg) on gestation day 9 resulted in anxiety-like behaviour specifically in male offspring, while no effects were detected in female offspring. We also found significant increases in the expression of GFAP and CNPase, as well as higher numbers of GFAP + astrocytes and O4+ oligodendrocytes in the prefrontal cortex of male offspring. Furthermore, increased scores for genes related to oligodendrocyte and lipid metabolism, particularly ApoE, were observed in the prefrontal cortex regions. Upon exposure to lipopolysaccharide during the ESC-to-neural stem cell (NSC) transition, Tuj1, Map2, Gfap, O4, and Oligo2 mRNA levels increased in the differentiated neural cells on day 14. In vitro experiments demonstrated that lipopolysaccharide exposure induced inflammatory responses, as evidenced by increased expression of IL1b and ApoB mRNA. Conclusions: Our findings suggest that prenatal infection at different stages of neural differentiation may result in distinct disturbances in neural differentiation during ESC—NSC transitions. Furthermore, early prenatal challenges with lipopolysaccharide selectively induce anxiety-like behaviour in male offspring. This behaviour may be attributed to the abnormal differentiation of astrocytes and oligodendrocytes in the brain, potentially mediated by ApoB/E signalling pathways in response to inflammatory stimuli. [ABSTRACT FROM AUTHOR]

Endogenous retroviruses (ERVs) are important components of transposable elements that constitute ∼40% of the mouse genome. ERVs exhibit dynamic expression patterns during early embryonic development and are engaged in numerous biological processes. Therefore, ERV expression must be closely monitored in cells. Most studies have focused on the regulation of ERV expression in mouse embryonic stem cells (ESCs) and during early embryonic development. This review touches on the classification, expression, and functions of ERVs in mouse ESCs and early embryos and mainly discusses ERV modulation strategies from the perspectives of transcription, epigenetic modification, nucleosome/chromatin assembly, and post-transcriptional control. [ABSTRACT FROM AUTHOR]

Tektins are a group of microtubule‐stabilizing proteins necessary for cilia and flagella assembly. TEKTIN1 (TEKT1) is used as a sperm marker for monitoring germ cell differentiation in embryonic stem (ES) and induced pluripotent stem (iPS) cells. Although upregulation of TEKT1 has been reported during spontaneous differentiation of ES and iPS cells, it is unclear which cells express TEKT1. To identify TEKT1‐expressing cells, we established an ES cell line derived from cynomolgus monkeys (Macaca fascicularis), which expresses Venus controlled by the TEKT1 promoter. Venus expression was detected at 5 weeks of differentiation on the surface of the embryoid body (EB), and it gradually increased with the concomitant formation of a leash‐like structure at the EB periphery. Motile cilia were observed on the surface of the Venus‐positive leash‐like structure after 8 weeks of differentiation. The expression of cilia markers as well as TEKT1–5 and 9 + 2 microtubule structures, which are characteristic of motile cilia, were detected in Venus‐positive cells. These results demonstrated that TEKT1‐expressing cells are multiciliated epithelial‐like cells that form a leash‐like structure during the spontaneous differentiation of ES and iPS cells. These findings will provide a new research strategy for studying cilia biology, including ciliogenesis and ciliopathies. [ABSTRACT FROM AUTHOR]

Summary: Stem cell technologies provide new opportunities for modeling cells in health and disease and for regenerative medicine. In both cases, developmental knowledge and defining the molecular properties and quality of the cell types is essential. In this study, we identify developmental factors important for the differentiation of human embryonic stem cells (hESCs) into functional midbrain dopaminergic (mDA) neurons. We found that laminin-511, and dual canonical and non-canonical WNT activation followed by GSK3β inhibition plus FGF8b, improved midbrain patterning. In addition, neurogenesis and differentiation were enhanced by activation of liver X receptors and inhibition of fibroblast growth factor signaling. Moreover, single-cell RNA-sequencing analysis revealed a developmental dynamics similar to that of the endogenous human ventral midbrain and the emergence of high-quality molecularly defined midbrain cell types, including mDA neurons. Our study identifies novel factors important for human midbrain development and opens the door for a future application of molecularly defined hESC-derived cell types in Parkinson disease.

Abstract Background Histone lactylation has been recently described as a novel histone post-translational modification linking cellular metabolism to epigenetic regulation. Results Given the expected relevance of this modification and current limited knowledge of its function, we generate genome-wide datasets of H3K18la distribution in various in vitro and in vivo samples, including mouse embryonic stem cells, macrophages, adipocytes, and mouse and human skeletal muscle. We compare them to profiles of well-established histone modifications and gene expression patterns. Supervised and unsupervised bioinformatics analysis shows that global H3K18la distribution resembles H3K27ac, although we also find notable differences. H3K18la marks active CpG island-containing promoters of highly expressed genes across most tissues assessed, including many housekeeping genes, and positively correlates with H3K27ac and H3K4me3 as well as with gene expression. In addition, H3K18la is enriched at active enhancers that lie in proximity to genes that are functionally important for the respective tissue. Conclusions Overall, our data suggests that H3K18la is not only a marker for active promoters, but also a mark of tissue specific active enhancers.

Due to the exquisite ability of cancer stemness to facilitate tumor initiation, metastasis, and cancer therapy resistance, targeting cancer stemness is expected to have clinical implications for cancer treatment. Genes are fundamental for forming and maintaining stemness. Considering shared genetic programs and pathways between embryonic stem cells and cancer stem cells, we conducted a study analyzing transcriptomic data of embryonic stem cells for mining potential cancer stemness genes. Firstly, we integrated co-expression and regression models and predicted 820 stemness genes. Results of gene enrichment analysis confirmed the good prediction performance for enriched signatures in cancer stem cells. Secondly, we provided an application case using the predicted stemness genes to construct a breast cancer stemness network. Mining on the network identified CD44, SOX2, TWIST1, and DLG4 as potential regulators of breast cancer stemness. Thirdly, using the signature of 31,028 chemical perturbations and their correlation with stemness marker genes, we predicted 67 stemness inhibitors with reasonable accuracy of 78%. Two drugs, namely Rigosertib and Proscillaridin A, were first identified as potential stemness inhibitors for melanoma and colon cancer, respectively. Overall, mining embryonic stem cell data provides a valuable way to identify cancer stemness regulators.

Background: Post-translational modifications of proteins are crucial to the regulation of their activity and function. As a newly discovered acylation modification, crotonylation of non-histone proteins remains largely unexplored, particularly in human embryonic stem cells (hESCs). Methods: We investigated the role of crotonylation in hESC differentiation by introduce crotonate into the culture medium of GFP tagged LTR7 primed H9 cell and extended pluripotent stem cell lines. RNA-seq assay was used to determine the hESC transcriptional features. Through morphological changes, qPCR of pluripotent and germ layer-specific gene markers and flow cytometry analysis, we determined that the induced crotonylation resulted in hESC differentiating into the endodermal lineage. We performed targeted metabolomic analysis and seahorse metabolic measurement to investigate the metabolism features after crotonate induction. Then high-resolution tandem mass spectrometry (LC–MS/MS) revealed the target proteins in hESCs. In addition, the role of crotonylated glycolytic enzymes (GAPDH and ENOA) was evaluated by in vitro crotonylation and enzymatic activity assays. Finally, we used knocked-down hESCs by shRNA, wild GAPDH and GAPDH mutants to explore potential role of GAPDH crotonylation in regulating human embryonic stem cell differentiation and metabolic switch. Result: We found that induced crotonylation in hESCs resulted in hESCs of different pluripotency states differentiating into the endodermal lineage. Increased protein crotonylation in hESCs was accompanied by transcriptomic shifts and decreased glycolysis. Large-scale crotonylation profiling of non-histone proteins revealed that metabolic enzymes were major targets of inducible crotonylation in hESCs. We further discovered GAPDH as a key glycolytic enzyme regulated by crotonylation during endodermal differentiation from hESCs. Conclusions: Crotonylation of GAPDH decreased its enzymatic activity thereby leading to reduced glycolysis during endodermal differentiation from hESCs. [ABSTRACT FROM AUTHOR]

Background: Three-dimensional (3D) human brain spheroids are instrumental to study central nervous system (CNS) development and (dys)function. Yet, in current brain spheroid models the limited variety of cell types hampers an integrated exploration of CNS (disease) mechanisms. Methods: Here we report a 5-month culture protocol that reproducibly generates H9 embryonic stem cell-derived human cortical spheroids (hCSs) with a large cell-type variety. Results: We established the presence of not only neuroectoderm-derived neural progenitor populations, mature excitatory and inhibitory neurons, astrocytes and oligodendrocyte (precursor) cells, but also mesoderm-derived microglia and endothelial cell populations in the hCSs via RNA-sequencing, qPCR, immunocytochemistry and transmission electron microscopy. Transcriptomic analysis revealed resemblance between the 5-months-old hCSs and dorsal frontal rather than inferior regions of human fetal brains of 19–26 weeks of gestational age. Pro-inflammatory stimulation of the generated hCSs induced a neuroinflammatory response, offering a proof-of-principle of the applicability of the spheroids. Conclusions: Our protocol provides a 3D human brain cell model containing a wide variety of innately developing neuroectoderm- as well as mesoderm-derived cell types, furnishing a versatile platform for comprehensive examination of intercellular CNS communication and neurological disease mechanisms. [ABSTRACT FROM AUTHOR]

Protein citrullination, including histone H1 and H3 citrullination, is important for transcriptional regulation, DNA damage response, and pluripotency of embryonic stem cells (ESCs). Tripartite motif containing 28 (Trim28), an embryonic development regulator involved in ESC self-renewal, has recently been identified as a novel substrate for citrullination by Padi4. However, the physiological functions of Trim28 citrullination and its role in regulating the chromatin structure and gene transcription of ESCs remain unknown. In this paper, we show that Trim28 is specifically citrullinated in mouse ESCs (mESCs), and that the loss of Trim28 citrullination induces loss of pluripotency. Mechanistically, Trim28 citrullination enhances the interaction of Trim28 with Smarcad1 and prevents chromatin condensation. Additionally, Trim28 citrullination regulates mESC pluripotency by promoting transcription of Nanog and Klf4 which it does by increasing the enrichment of H3K27ac and H3K4me3 and decreasing the enrichment of H3K9me3 in the transcriptional regulatory region. Thus, our findings suggest that Trim28 citrullination is the key for the epigenetic activation of pluripotency genes and pluripotency maintenance of ESCs. Together, these results uncover a role Trim28 citrullination plays in pluripotency regulation and provide novel insight into how citrullination of proteins other than histones regulates chromatin compaction. [ABSTRACT FROM AUTHOR]

This research was conducted to investigate the growth performance of black-boned chicken embryonic stem (ES) cells for the future development of cultured meat. Black-boned chicken ES cells were isolated and cultured from fertilized eggs. The treatments applied were: fetal bovine serum (FBS) (T1), commercial chicken serum (SCK) (T2), Pradu Hang Dam chicken serum (PDC) (T3), and black-boned chicken serum (BBC) (T4). Black-boned chicken ES cells were cultured at 37.0 °C in a humidified environment of 5% CO2, for 10 days. The growth of black-boned chicken ES cells concentration was measured by the absorbance at 450 nm. A haemocytometer was used to count the number of black-boned chicken ES cells. Comparing the protein content of cultured meat and chicken meat was collected for combustion and proximate analysis. All collected data were analyzed using ANOVA in a completely randomized design. T4 tended to have a higher number and growth rate than the other groups, followed by T3, T2, and T1, respectively. When counted ES cells final, T4 had a significantly higher number and growth rate than the other groups (p

Dynamic-related protein 1 (DRP1) is a key protein of mitochondrial fission. In this study, we found that inhibition of activity of DRP1 led to increased levels of cleavage embryo genes in mouse embryonic stem cells (mESCs), which might reflect a transient totipotency status derived from pluripotency. This result indicates that DRP1 inhibition in mESCs leads to a tendency to obtain a new expression profile similar to that of the 2C-like state. Meanwhile, we also noticed that the glycolysis/gluconeogenesis pathway and its related enzymes were significantly downregulated, and the key glycolytic enzymes were also downregulated in various 2C-like cells. Moreover, when DRP1 activity was inhibited from the late zygote when cleavage embryo genes started to express, development of early embryos was inhibited, and these cleavage embryo genes failed to be efficiently silenced at the late 2-cell (2C) stage. Taken together, our result shows that DRP1 plays an important role in silencing cleavage embryo genes for totipotency-to-pluripotency transition.

Cell identity is determined by the transcriptional regulation of a cell‐type‐specific gene group. The Paf1 complex (Paf1C), an RNA polymerase II‐associating factor, is an important transcriptional regulator that not only participates in transcription elongation and termination but also affects transcription‐coupled histone modifications and chromatin organisation. Recent studies have shown that Paf1C is involved in the expression of genes required for self‐renewal and pluripotency in stem cells and tumorigenesis. In this review, we focused on the role of Paf1C as a critical transcriptional regulator in cell fate decisions. Paf1C affects the pluripotency of stem cells by regulating the expression of core transcription factors such as Oct4 and Nanog. In addition, Paf1C directly binds to the promoters or distant elements of target genes, thereby maintaining the pluripotency in embryonic stem cells derived from an early stage of the mammalian embryo. Paf1C is upregulated in cancer stem cells, as compared with that in cancer cells, suggesting that Paf1C may be a target for cancer therapy. Interestingly, Paf1C is involved in multiple developmental stages in Drosophila, zebrafish, mice and even humans, thereby displaying a trend for the correlation between Paf1C and cell fate. Thus, we propose that Paf1C is a critical contributor to cell differentiation, cell specification and its characteristics and could be employed as a therapeutic target in developmental diseases. [ABSTRACT FROM AUTHOR]

Congenital hydrocephalus (CH) is a major cause of childhood morbidity. Mono‐allelic mutations in Trim71, a conserved stem‐cell‐specific RNA‐binding protein, cause CH; however, the molecular basis for pathogenesis mediated by these mutations remains unknown. Here, using mouse embryonic stem cells as a model, we reveal that the mouse R783H mutation (R796H in human) alters Trim71's mRNA substrate specificity and leads to accelerated stem‐cell differentiation and neural lineage commitment. Mutant Trim71, but not wild‐type Trim71, binds Lsd1 (Kdm1a) mRNA and represses its translation. Specific inhibition of this repression or a slight increase of Lsd1 in the mutant cells alleviates the defects in stem cell differentiation and neural lineage commitment. These results determine a functionally relevant target of the CH‐causing Trim71 mutant that can potentially be a therapeutic target and provide molecular mechanistic insights into the pathogenesis of this disease. Synopsis: A congenital‐hydrocephalus‐causing mutation of the RNA‐binding protein Trim71 alters its mRNA substrates. Mutant but not wild‐type Trim71 represses Lsd1 expression and induces stem cell differentiation defects. A congenital‐hydrocephalus‐causing mutation of the RNA‐binding protein Trim71 alters, but does not abolish, its RNA‐binding ability.Mutant Trim71 represses Lsd1 mRNA translation and induces differentiation defects in embryonic stem cells. [ABSTRACT FROM AUTHOR]

Despite a prominent risk factor for Neurodevelopmental disorders (NDD), it remains unclear how Autism Susceptibility Candidate 2 (AUTS2) controls the neurodevelopmental program. Our studies investigated the role of AUTS2 in neuronal differentiation and discovered that AUTS2, together with WDR68 and SKI, forms a novel protein complex (AWS) specifically in neuronal progenitors and promotes neuronal differentiation through inhibiting BMP signaling. Genomic and biochemical analyses demonstrated that the AWS complex achieves this effect by recruiting the CUL4 E3 ubiquitin ligase complex to mediate poly-ubiquitination and subsequent proteasomal degradation of phosphorylated SMAD1/5/9. Furthermore, using primary cortical neurons, we observed aberrant BMP signaling and dysregulated expression of neuronal genes upon manipulating the AWS complex, indicating that the AWS-CUL4-BMP axis plays a role in regulating neuronal lineage specification in vivo. Thus, our findings uncover a sophisticated cellular signaling network mobilized by a prominent NDD risk factor, presenting multiple potential therapeutic targets for NDD. [ABSTRACT FROM AUTHOR]