The Presence or Absence of Alkaline Phosphatase Activity to Discriminate Pluripotency Characteristics in Porcine Epiblast Stem Cell-Like Cells.

Porcine embryonic stem cells (pESCs) would provide potentials for agricultural- and biotechnological-related applications. However, authentic pESCs have not been established yet because standards for porcine stem cell-specific markers and culture conditions are not clear. Therefore, the present study reports attempts to derive pluripotent epiblast stem cells either from in vitro or in vivo derived porcine embryos. Nine epiblast cell lines (seven lines from Berkshire and two lines from Duroc) could only be isolated from day 9- to 9.5-old in vivo derived early conceptuses. Pluripotency features were analyzed in relation to the presence or absence of alkaline phosphatase (AP) activity. Interestingly, the mRNA expression of several marker genes for pluripotency or epiblast was different between putative epiblast stem cells of the two groups [AP-positive (+) pEpiSC-like cell 2 line and AP-negative (-) pEpiSC-like cell 8 line]. For example, expressions of OCT-3/4, NANOG, SOX2, c-MYC, FGF2, and NODAL in AP-negative (-) porcine epiblast stem cell (pEpiSC)-like cells were higher than those in AP-positive (+) pEpiSC-like cells. Expression of surface markers differed between the two groups to some extent. SSEA-1 was strongly expressed only in AP-negative (-) pEpiSC-like cells, whereas AP-positive (+) pEpiSC-like cells did not express. In addition, we report to have some differences in the in vitro differentiation capacity between AP-positive (+) and AP-negative (-) epiblast cell lines. Primary embryonic germ layer markers (cardiac actin, nestin, and GATA 6) and primordial germ cell markers (Dazl and Vasa) were strongly expressed in embryoid bodies (EBs) aggregated from AP-negative (-) pEpiSC-like cells, whereas EBs aggregated from AP-positive (+) pEpiSCs did not show expression of primary embryonic germ layers and primordial germ cell markers except GATA 6. These results indicate that pEpiSC-like cells display different pluripotency characteristics in relation to AP activity.

Ubiquitin-Specific Protease 3 Deubiquitinates and Stabilizes Oct4 Protein in Human Embryonic Stem Cells.

Oct4 is an important mammalian POU family transcription factor expressed by early human embryonic stem cells (hESCs). The precise level of Oct4 governs the pluripotency and fate determination of hESCs. Several post-translational modifications (PTMs) of Oct4 including phosphorylation, ubiquitination, and SUMOylation have been reported to regulate its critical functions in hESCs. Ubiquitination and deubiquitination of Oct4 should be well balanced to maintain the pluripotency of hESCs. The protein turnover of Oct4 is regulated by several E3 ligases through ubiquitin-mediated degradation. However, reversal of ubiquitination by deubiquitinating enzymes (DUBs) has not been reported for Oct4. In this study, we generated a ubiquitin-specific protease 3 (USP3) gene knockout using the CRISPR/Cas9 system and demonstrated that USP3 acts as a protein stabilizer of Oct4 by deubiquitinating Oct4. USP3 interacts with endogenous Oct4 and co-localizes in the nucleus of hESCs. The depletion of USP3 leads to a decrease in Oct4 protein level and loss of pluripotent morphology in hESCs. Thus, our results show that USP3 plays an important role in controlling optimum protein level of Oct4 to retain pluripotency of hESCs.

Stromal DLK1 promotes proliferation and inhibits differentiation of the intestinal epithelium during development.

The stem/progenitor cells of the developing intestine are biologically distinct from their adult counterparts. Here, we examine the microenvironmental cues that regulate the embryonic stem/progenitor population, focusing on the role of Notch pathway factor delta-like protein-1 (DLK1). mRNA-seq analyses of intestinal mesenchymal cells (IMCs) collected from embryonic day 14.5 (E14.5) or adult IMCs and a novel coculture system with E14.5 intestinal epithelial organoids were used. Following addition of recombinant DLK1 (rDLK) or Dlk1 siRNA (siDlk1), epithelial characteristics were compared using imaging, replating efficiency assays, qPCR, and immunocytochemistry. The intestinal phenotypes of littermate Dlk1+/+ and Dlk1-/- mice were compared using immunohistochemistry. Using transcriptomic analyses, we identified morphogens derived from the embryonic mesenchyme that potentially regulate the developing epithelial cells, to focus on Notch family candidate DLK1. Immunohistochemistry indicated that DLK1 was expressed exclusively in the intestinal stroma at E14.5 at the top of emerging villi, decreased after birth, and shifted to the intestinal epithelium in adulthood. In coculture experiments, addition of rDLK1 to adult IMCs inhibited organoid differentiation, whereas Dlk1 knockdown in embryonic IMCs increased epithelial differentiation to secretory lineage cells. Dlk1-/- mice had restricted Ki67+ cells in the villi base and increased secretory lineage cells compared with Dlk1+/+ embryos. Mesenchyme-derived DLK1 plays an important role in the promotion of epithelial stem/precursor expansion and prevention of differentiation to secretory lineages in the developing intestine.NEW & NOTEWORTHY Using a novel coculture system, transcriptomics, and transgenic mice, we investigated differential molecular signaling between the intestinal epithelium and mesenchyme during development and in the adult. We show that the Notch pathway factor delta-like protein-1 (DLK1) is stromally produced during development and uncover a new role for DLK1 in the regulation of intestinal epithelial stem/precursor expansion and differentiation to secretory lineages.

Long Non-coding RNA Depletion Using Self-Cleaving Ribozymes.

Some long non-coding RNA (lncRNA) genes encode a functional RNA product, whereas others act as DNA elements or via the act of transcription . We describe here a ribozyme-based approach to deplete an endogenous lncRNA in mouse embryonic stem cells, with minimal disruption of its gene. This enables the role of the lncRNA product to be tested.

Complex functions of Gcn5 and Pcaf in development and disease.

A wealth of biochemical and cellular data, accumulated over several years by multiple groups, has provided a great degree of insight into the molecular mechanisms of actions of GCN5 and PCAF in gene activation. Studies of these lysine acetyltransferases (KATs) in vitro, in cultured cells, have revealed general mechanisms for their recruitment by sequence-specific binding factors and their molecular functions as transcriptional co-activators. Genetic studies indicate that GCN5 and PCAF are involved in multiple developmental processes in vertebrates, yet our understanding of their molecular functions in these contexts remains somewhat rudimentary. Understanding the functions of GCN5/PCAF in developmental processes provides clues to the roles of these KATs in disease states. Here we will review what is currently known about the developmental roles of GCN5 and PCAF, as well as emerging role of these KATs in oncogenesis.

Regulation and Effect of Telomerase and Telomeric Length in Stem Cells.

Telomeres are the protective end caps of eukaryotic chromosomes and they determine the proliferative lifespan of somatic cells, as the protectors of cell replication. Telomere length in leucocytes reflects telomere length in other somatic cells. Leucocyte telomere length can be a biomarker of human ageing. The risk of diseases associated with reduced cell proliferation and tissue degeneration, including aging or aging-associated diseases, such as dyskeratosis congenita, cardiovascular diseases, pulmonary fibrosis and aplastic anemia, is correlated with an increase in the shortening of telomeres. On the other hand, the risk of diseases that are associated with increased proliferative growth, including major cancers, is correlated with long telomeres. In most of the cancers, a telomere maintenance mechanism during DNA replication is essential. The reactivation of the functional ribonucleoprotein holoenzyme complex (telomerase) starts the cascade from normal and premalignant somatic cells to advanced malignant cells. Telomerase is overexpressed during the development of cancer and embryonic stem cells, through controlling genome integrity, cancer formation and stemness. Cancer cells have mechanisms to maintain telomeres to avoid initiation of cellular senescence or apoptosis, and halting cell division by critically short telomeres. Modulation of the human telomerase reverse transcriptase is the rate-limiting step for the production of functional telomerase and telomere maintenance. The human telomerase reverse transcriptase promoter promotes its gene expression only in tumor cells, but not in normal cells. Some cancers activate an alternative expansion of telomeres maintenance mechanism via DNA recombination to reduce the shortening of their telomeres. Not only heritability but also oxidative stress, inflammation, environmental factors, and therapeutic interventions have an effect on telomere shortening, explaining the variability in telomere length across individuals. There have been a large number of publications, which correlate human diseases with progressive telomere shortening. Telomere length of an individual at birth is also important to follow up telomere shortening, and it can be used as a biomarker for healthy aging. On the other hand, understanding of cellular stress factors, which affect stem cell behavior, will be useful in regeneration or treatment of cancer and age-associated diseases. In this review, we will understand the connection between stem cell and telomere biology, cancer, and aging-associated diseases. This connection may be useful for discovering novel drug targets and improve outcomes for patients having cancer and aging-associated diseases.

Embryonic Stem Cell-Derived Extracellular Vesicles Maintain ESC Stemness by Activating FAK.

It is critical that epiblast cells within blastocyst-stage embryos receive the necessary regulatory cues to remain pluripotent until the appropriate time when they are stimulated to undergo differentiation, ultimately to give rise to an entire organism. Here, we show that exposure of embryonic stem cells (ESCs), which are the in vitro equivalents of epiblasts, to ESC-derived extracellular vesicles (EVs) helps to maintain their stem cell properties even under culture conditions that would otherwise induce differentiation. EV-treated ESCs continued to express stemness genes, preserving their pluripotency and ability to generate chimeric mice. These effects were triggered by fibronectin bound to the surfaces of EVs, enabling them to interact with ESC-associated integrins and activate FAK more effectively than fibronectin alone. Overall, these findings highlight a potential regulatory mechanism whereby epiblast cells, via their shed EVs, create an environment within the blastocyst that prevents their premature differentiation and maintains their pluripotent state.

ETV2 (Ets Variant Transcription Factor 2)-Rhoj Cascade Regulates Endothelial Progenitor Cell Migration During Embryogenesis.

OBJECTIVE: Endothelial progenitors migrate early during embryogenesis to form the primary vascular plexus. The regulatory mechanisms that govern their migration are not completely defined. Here, we describe a novel role for ETV2 (Ets variant transcription factor 2) in cell migration and provide evidence for an ETV2-Rhoj network as a mechanism responsible for this process. Approach and Results: Analysis of RNAseq datasets showed robust enrichment of migratory/motility pathways following overexpression of ETV2 during mesodermal differentiation. We then analyzed ETV2 chromatin immunoprecipitation-seq and assay for transposase accessible chromatin-seq datasets, which showed enrichment of chromatin immunoprecipitation-seq peaks with increased chromatin accessibility in migratory genes following overexpression of ETV2. Migratory assays showed that overexpression of ETV2 enhanced cell migration in mouse embryonic stem cells, embryoid bodies, and mouse embryonic fibroblasts. Knockout of Etv2 led to migratory defects of Etv2-EYFP+ angioblasts to their predefined regions of developing embryos relative to wild-type controls at embryonic day (E) 8.5, supporting its role during migration. Mechanistically, we showed that ETV2 binds the promoter region of Rhoj serving as an upstream regulator of cell migration. Single-cell RNAseq analysis of Etv2-EYFP+ sorted cells revealed coexpression of Etv2 and Rhoj in endothelial progenitors at E7.75 and E8.25. Overexpression of ETV2 led to a robust increase in Rhoj in both embryoid bodies and mouse embryonic fibroblasts, whereas, its expression was abolished in the Etv2 knockout embryoid bodies. Finally, shRNA-mediated knockdown of Rhoj resulted in migration defects, which were partially rescued by overexpression of ETV2. CONCLUSIONS: These results define an ETV2-Rhoj cascade, which is important for the regulation of endothelial progenitor cell migration.

Distinct contributions of DNA methylation and histone acetylation to the genomic occupancy of transcription factors.

Epigenetic modifications on chromatin play important roles in regulating gene expression. Although chromatin states are often governed by multilayered structure, how individual pathways contribute to gene expression remains poorly understood. For example, DNA methylation is known to regulate transcription factor binding but also to recruit methyl-CpG binding proteins that affect chromatin structure through the activity of histone deacetylase complexes (HDACs). Both of these mechanisms can potentially affect gene expression, but the importance of each, and whether these activities are integrated to achieve appropriate gene regulation, remains largely unknown. To address this important question, we measured gene expression, chromatin accessibility, and transcription factor occupancy in wild-type or DNA methylation-deficient mouse embryonic stem cells following HDAC inhibition. We observe widespread increases in chromatin accessibility at retrotransposons when HDACs are inhibited, and this is magnified when cells also lack DNA methylation. A subset of these elements has elevated binding of the YY1 and GABPA transcription factors and increased expression. The pronounced additive effect of HDAC inhibition in DNA methylation-deficient cells demonstrates that DNA methylation and histone deacetylation act largely independently to suppress transcription factor binding and gene expression.

Cops5 safeguards genomic stability of embryonic stem cells through regulating cellular metabolism and DNA repair.

The highly conserved COP9 signalosome (CSN), composed of 8 subunits (Cops1 to Cops8), has been implicated in pluripotency maintenance of human embryonic stem cells (ESCs). Yet, the mechanism for the CSN to regulate pluripotency remains elusive. We previously showed that Cops2, independent of the CSN, is essential for the pluripotency maintenance of mouse ESCs. In this study, we set out to investigate how Cops5 and Cops8 regulate ESC differentiation and tried to establish Cops5 and Cops8 knockout (KO) ESC lines by CRISPR/Cas9. To our surprise, no Cops5 KO ESC clones were identified out of 127 clones, while three Cops8 KO ESC lines were established out of 70 clones. We then constructed an inducible Cops5 KO ESC line. Cops5 KO leads to decreased expression of the pluripotency marker Nanog, proliferation defect, G2/M cell-cycle arrest, and apoptosis of ESCs. Further analysis revealed dual roles of Cops5 in maintaining genomic stability of ESCs. On one hand, Cops5 suppresses the autophagic degradation of Mtch2 to direct cellular metabolism toward glycolysis and minimize reactive oxygen species (ROS) production, thereby reducing endogenous DNA damage. On the other hand, Cops5 is required for high DNA damage repair (DDR) activities in ESCs. Without Cops5, elevated ROS and reduced DDR activities lead to DNA damage accumulation in ESCs. Subsequently, p53 is activated to trigger G2/M arrest and apoptosis. Altogether, our studies reveal an essential role of Cops5 in maintaining genome integrity and self-renewal of ESCs by regulating cellular metabolism and DDR pathways.

A novel member of Prame family, Gm12794c, counteracts retinoic acid differentiation through the methyltransferase activity of PRC2.

Embryonic stem cells (ESCs) fluctuate among different levels of pluripotency defined as metastates. Sporadically, metastable cellular populations convert to a highly pluripotent metastate that resembles the preimplantation two-cell embryos stage (defined as 2C stage) in terms of transcriptome, DNA methylation, and chromatin structure. Recently, we found that the retinoic acid (RA) signaling leads to a robust increase of cells specifically expressing 2C genes, such as members of the Prame family. Here, we show that Gm12794c, one of the most highly upregulated Prame members, and previously identified as a key player for the maintenance of pluripotency, has a functional role in conferring ESCs resistance to RA signaling. In particular, RA-dependent expression of Gm12794c induces a ground state-like metastate, as evaluated by activation of 2C-specific genes, global DNA hypomethylation and rearrangement of chromatin similar to that observed in naive totipotent preimplantation epiblast cells and 2C-like cells. Mechanistically, we demonstrated that Gm12794c inhibits Cdkn1A gene expression through the polycomb repressive complex 2 (PRC2) histone methyltransferase activity. Collectively, our data highlight a molecular mechanism employed by ESCs to counteract retinoic acid differentiation stimuli and contribute to shed light on the molecular mechanisms at grounds of ESCs naive pluripotency-state maintenance.

PRC1 Catalytic Activity Is Central to Polycomb System Function.

The Polycomb repressive system is an essential chromatin-based regulator of gene expression. Despite being extensively studied, how the Polycomb system selects its target genes is poorly understood, and whether its histone-modifying activities are required for transcriptional repression remains controversial. Here, we directly test the requirement for PRC1 catalytic activity in Polycomb system function. To achieve this, we develop a conditional mutation system in embryonic stem cells that completely removes PRC1 catalytic activity. Using this system, we demonstrate that catalysis by PRC1 drives Polycomb chromatin domain formation and long-range chromatin interactions. Furthermore, we show that variant PRC1 complexes with DNA-binding activities occupy target sites independently of PRC1 catalytic activity, providing a putative mechanism for Polycomb target site selection. Finally, we discover that Polycomb-mediated gene repression requires PRC1 catalytic activity. Together these discoveries provide compelling evidence that PRC1 catalysis is central to Polycomb system function and gene regulation.

Fetal and trophoblast PI3K p110α have distinct roles in regulating resource supply to the growing fetus in mice.

Studies suggest that placental nutrient supply adapts according to fetal demands. However, signaling events underlying placental adaptations remain unknown. Here we demonstrate that phosphoinositide 3-kinase p110α in the fetus and the trophoblast interplay to regulate placental nutrient supply and fetal growth. Complete loss of fetal p110α caused embryonic death, whilst heterozygous loss resulted in fetal growth restriction and impaired placental formation and nutrient transport. Loss of trophoblast p110α resulted in viable fetuses, abnormal placental development and a failure of the placenta to transport sufficient nutrients to match fetal demands for growth. Using RNA-seq we identified genes downstream of p110α in the trophoblast that are important in adapting placental phenotype. Using CRISPR/Cas9 we showed loss of p110α differentially affects gene expression in trophoblast and embryonic stem cells. Our findings reveal important, but distinct roles for p110α in the different compartments of the conceptus, which control fetal resource acquisition and growth.

Electrochemical Imaging of Cell Activity in Hydrogels Embedded in Grid-shaped Polycaprolactone Scaffolds Using a Large-scale Integration-based Amperometric Device.

Tissue engineering requires analytical methods to monitor cell activity in hydrogels. Here, we present a method for the electrochemical imaging of cell activity in hydrogels embedded in printed polycaprolactone (PCL) scaffolds. Because a structure made of only hydrogel is fragile, PCL frameworks are used as a support material. A grid-shaped PCL was fabricated using an excluder printer. Photocured hydrogels containing cells were set at each grid hole, and cell activity was monitored using a large-scale integration-based amperometric device. The electrochemical device contains 400 microelectrodes for biomolecule detection, such as dissolved oxygen and enzymatic products. As proof of the concept, alkaline phosphatase and respiration activities of embryonic stem cells in the hydrogels were electrochemically monitored. The results indicate that the electrochemical imaging is useful for evaluating cells in printed scaffolds.

Reduced MEK inhibition preserves genomic stability in naive human embryonic stem cells.

Human embryonic stem cells (hESCs) can be captured in a primed state in which they resemble the postimplantation epiblast, or in a naive state where they resemble the preimplantation epiblast. Naive-cell-specific culture conditions allow the study of preimplantation development ex vivo but reportedly lead to chromosomal abnormalities, which compromises their utility in research and potential therapeutic applications. Although MEK inhibition is essential for the naive state, here we show that reduced MEK inhibition facilitated the establishment and maintenance of naive hESCs that retained naive-cell-specific features, including global DNA hypomethylation, HERVK expression, and two active X chromosomes. We further show that hESCs cultured under these modified conditions proliferated more rapidly; accrued fewer chromosomal abnormalities; and displayed changes in the phosphorylation levels of MAPK components, regulators of DNA damage/repair, and cell cycle. We thus provide a simple modification to current methods that can enable robust growth and reduced genomic instability in naive hESCs.

Superoxide dismutase 1 expression is modulated by the core pluripotency transcription factors Oct4, Sox2 and Nanog in embryonic stem cells.

Redox homeostasis is vital for cellular functions and to prevent the detrimental consequences of oxidative stress. Pluripotent stem cells (PSCs) have an enhanced antioxidant system which supports the preservation of their genome. Besides, reactive oxygen species (ROS) are proposed to be involved in both self-renewal maintenance and in differentiation in embryonic stem cells (ESCs). Increasing evidence shows that cellular systems related to the oxidative stress defense decline along differentiation of PSCs. Although redox homeostasis has been extensively studied for many years, the knowledge about the transcriptional regulation of the genes involved in these systems is still limited. In this work, we studied Sod1 gene modulation by the PSCs fundamental transcription factors Oct4, Sox2 and Nanog. We found that this gene, which is expressed in mouse ESCs (mESCs), was repressed when they were induced to differentiate. Accordingly, these factors induced Sod1 promoter activity in a trans-activation assay. Finally, Sod1 mRNA levels were reduced when Oct4, Sox2 and Nanog were down-regulated by a shRNA approach in mESCs. Taken together, we found that PSCs' key transcription factors are involved in the modulation of Sod1 gene, suggesting a relationship between the pluripotency core and redox homeostasis in these cells.

Genome-wide tracking of dCas9-methyltransferase footprints.

In normal mammalian development cytosine methylation is essential and is directed to specific regions of the genome. Despite notable advances through mapping its genome-wide distribution, studying the direct contribution of DNA methylation to gene and genome regulation has been limited by the lack of tools for its precise manipulation. Thus, combining the targeting capability of the CRISPR-Cas9 system with an epigenetic modifier has attracted interest in the scientific community. In contrast to profiling the genome-wide cleavage of a nuclease competent Cas9, tracing the global activity of a dead Cas9 (dCas9) methyltransferase fusion protein is challenging within a highly methylated genome. Here, we report the generation and use of an engineered, methylation depleted but maintenance competent mouse ES cell line and find surprisingly ubiquitous nuclear activity of dCas9-methyltransferases. Subsequent experiments in human somatic cells refine these observations and point to an important difference between genetic and epigenetic editing tools that require unique experimental considerations.

miR-124 and miR-9 mediated downregulation of HDAC5 promotes neurite development through activating MEF2C-GPM6A pathway.

The class IIa histone deacetylases (HDACs) play important roles in the central nervous system during diverse biological processes such as synaptic plasticity, axon regeneration, cell apoptosis, and neural differentiation. Although it is known that HDAC5 regulates neuronal differentiation, neither the physiological function nor the regulation of HDAC5 in neuronal differentiation is clear. Here, we identify HDAC5 as an inhibitor of neurite elongation and show that HDAC5 is regulated by the brain enriched microRNA miR-124 and miR-9. We discover that HDAC5 inhibits neurite extension both in differentiated P19 cells and primary neurons. We also show that the neuronal membrane glycoprotein GPM6A (M6a) is a direct target gene of HDAC5 regulated transcriptional factor MEF2C. HDAC5 inhibits neurite elongation, acting at least partially via a MEF2C/M6a signaling pathway. We also confirmed the miR-124/miR-9 regulated HDAC5-MEF2C-M6a pathway regulates neurite development in primary neurons. Thus, HDAC5 emerges as a cellular conductor of MEF2C and M6a activity and is regulated by miR-124 and miR-9 to control neurite development.

Phosphoproteomics links glycogen synthase kinase-3 to RNA splicing.

Protein kinases play essential biological roles by phosphorylating a diverse range of signaling molecules, but deciphering their direct physiological targets remains a challenge. A new study by Shinde et al. uses phosphoproteomics to identify glycogen synthase kinase-3 (GSK-3) substrates in mouse embryonic stem cells (mESCs), providing a broad profile of GSK-3 activity and defining a new role for this central kinase in regulating RNA splicing.

Aurora Kinase B, a novel regulator of TERF1 binding and telomeric integrity.

AURKB (Aurora Kinase B) is a serine/threonine kinase better known for its role at the mitotic kinetochore during chromosome segregation. Here, we demonstrate that AURKB localizes to the telomeres in mouse embryonic stem cells, where it interacts with the essential telomere protein TERF1. Loss of AURKB function affects TERF1 telomere binding and results in aberrant telomere structure. In vitro kinase experiments successfully identified Serine 404 on TERF1 as a putative AURKB target site. Importantly, in vivo overexpression of S404-TERF1 mutants results in fragile telomere formation. These findings demonstrate that AURKB is an important regulator of telomere structural integrity.