Pluripotency Of Mouse Embryonic Stem Cells Research Articles

The pluripotent-to-totipotent state transition in mESCs activates the intrinsic apoptotic pathway through DUX-induced DNA replication stress

The pluripotent mouse embryonic stem cell (mESCs) can transit into the totipotent-like state, and the transcription factor DUX is one of the master regulators of this transition. Intriguingly, this transition in mESCs is accompanied by massive cell death, which significantly impedes the establishment and maintenance of totipotent cells in vitro, yet the underlying mechanisms of this cell death remain largely elusive. In this study, we found that the totipotency transition in mESCs triggered cell death through the upregulation of DUX. Specifically, R-loops are accumulated upon DUX induction, which subsequently lead to DNA replication stress (RS) in mESCs. This RS further activates p53 and PMAIP1, ultimately leading to Caspase-9/7-dependent intrinsic apoptosis. Notably, inhibiting this intrinsic apoptosis not only mitigates cell death but also enhances the efficiency of the totipotency transition in mESCs. Our findings thus elucidate one of the mechanisms underlying cell apoptosis during the totipotency transition in mESCs and provide a strategy for optimizing the establishment and maintenance of totipotent cells in vitro.

Multimodal screen identifies noise-regulatory proteins.

Gene-expression noise can influence cell-fate choices across pathology and physiology. However, a crucial question persists: do regulatory proteins or pathways exist that control noise independently of mean expression levels? Our integrative approach, combining single-cell RNA sequencing with proteomics and regulator enrichment analysis, identifies 32 putative noise regulators. SON, a nuclear speckle-associated protein, alters transcriptional noise without changing mean expression levels. Furthermore, SON's noise control can propagate to the protein level. Long-read and total RNA sequencing shows that SON's noise control does not significantly change isoform usage or splicing efficiency. Moreover, SON depletion reduces state switching in pluripotent mouse embryonic stem cells and impacts their fate choice during differentiation. Collectively, we demonstrate a class of proteins that control noise orthogonally to mean expression levels. This work serves as a proof of concept that can identify other functional noise regulators throughout development and disease progression.

M6A mRNA methylation by METTL14 regulates early pancreatic cell differentiation.

N6-methyladenosine (m6A) is the most abundant chemical modification in mRNA and plays important roles in human and mouse embryonic stem cell pluripotency, maintenance, and differentiation. We have recently reported that m6A is involved in the postnatal control of β-cell function in physiological states and in type 1 and 2 diabetes. However, the precise mechanisms by which m6A acts to regulate the development of human and mouse pancreas are unexplored. Here, we show that the m6A landscape is dynamic during human pancreas development, and that METTL14, one of the m6A writer complex proteins, is essential for the early differentiation of both human and mouse pancreatic cells.

Identification of an embryonic differentiation stage marked by Sox1 and FoxA2 co-expression using combined cell tracking and high dimensional protein imaging

Pluripotent mouse embryonic stem cells (ESCs) can differentiate to all germ layers and serve as an in vitro model of embryonic development. To better understand the differentiation paths traversed by ESCs committing to different lineages, we track individual differentiating ESCs by timelapse imaging followed by multiplexed high-dimensional Imaging Mass Cytometry (IMC) protein quantification. This links continuous live single-cell molecular NANOG and cellular dynamics quantification over 5-6 generations to protein expression of 37 different molecular regulators in the same single cells at the observation endpoints. Using this unique data set including kinship history and live lineage marker detection, we show that NANOG downregulation occurs generations prior to, but is not sufficient for neuroectoderm marker Sox1 upregulation. We identify a developmental cell type co-expressing both the canonical Sox1 neuroectoderm and FoxA2 endoderm markers in vitro and confirm the presence of such a population in the post-implantation embryo. RNASeq reveals cells co-expressing SOX1 and FOXA2 to have a unique cell state characterized by expression of both endoderm as well as neuroectoderm genes suggesting lineage potential towards both germ layers.

Unveiling the mystery of nuclear RNA homeostasis

How nuclear RNA homeostasis impacts cellular functions remains elusive. In this issue of Cell Stem Cell, Han etal.1 utilized a controllable protein degradation system targeting EXOSC2 to perturb RNA homeostasis in mouse pluripotent embryonic stem cells, revealing its vital role in orchestrating crucial nuclear events for cellular fitness.

Dynamic Roles of Signaling Pathways in Maintaining Pluripotency of Mouse and Human Embryonic Stem Cells.

Culturing of mouse and human embryonic stem cells (ESCs) in vitro was a major breakthrough in the field of stem cell biology. These models gained popularity very soon mainly due to their pluripotency. Evidently, the ESCs of mouse and human origin share typical phenotypic responses due to their pluripotent nature, such as self-renewal capacity and potency. The conserved network of core transcription factors regulates these responses. However, significantly different signaling pathways and upstream transcriptional networks regulate expression and activity of these core pluripotency factors in ESCs of both the species. In fact, ample evidence shows that a pathway, which maintains pluripotency in mouse ESCs, promotes differentiation in human ESCs. In this review, we discuss the role of canonical signaling pathways implicated in regulation of pluripotency and differentiation particularly in mouse and human ESCs. We believe that understanding these distinct and at times-opposite mechanisms-is critical for the progress in the field of stem cell biology and regenerative medicine.

Enhancing the neural differentiation capabilities of genetically asymmetric mouse F1 hybrid embryonic stem cell lines.

Allele-specific, monoallelic expression in diploid organisms represents an extreme case of allelic imbalance resulting from incompatibility between cis- and trans-elements. Due to haploinsufficiency, such monoallelic expression can lead to sporadic genetic diseases. In mice, allelic imbalances can be introduced into F1 offspring from inbred strains. Previously, we established F1 hybrid embryonic stem (ES) cell lines derived from four different mouse strains, each belonging to a different subspecies with substantial genetic polymorphisms. In this study, we investigated the neural differentiation capacity of the established ES cell lines. By introducing different culture conditions, which kept the ES cells undifferentiated under various pluripotencies, we succeeded in differentiating the majority of ES cell lines (eight out of eleven) with our default neural differentiation paradigm. Still, three lines exhibited insufficient differentiation despite combining culture conditions promoting undifferentiated as well as differentiated status. In addition, Ube3a imprinting was seen in two lines. Our findings contribute to the methodological understanding of mouse ES cell pluripotency and lead to the practical utility of F1 hybrid ES cells as a model for studying phenotypes resulting from gene locus interactions.

Dissecting the Role of the Ccr4-Not Deadenylase Complex in Pluripotency and Differentiation.

In this chapter, we describe methods and assays to examine the role of the Ccr4-Not mRNA deadenylase complex in regulating mouse embryonic stem cell (ESC) pluripotency and differentiation. We present the following procedures: sgRNA design and cloning, the culture of mouse ESCs, transfection and colony picking, genotyping, generation of Cnot3 conditional deletion ESCs, assessment of Cnot3 deletion efficiency, and examination of the impact on ESC maintenance and differentiation. The above approach and protocols can also be readily applied to study other Ccr4-Not subunits in ESC fate regulation, thereby facilitating the systematic dissection of Ccr4-Not function in stem cells and development.

Histone proteomics implicates H3K36me2 and its regulators in mouse embryonic stem cell pluripotency exit and lineage choice

Abstract Objectives Gene expression changes during embryonic stem cell (ESC) differentiation is regulated by epigenetic mechanisms. Understanding these can help uncover how cell fate decisions are made during early embryonic development. Comparison of chromatin of ESCs with lineage-committed cells can implicate chromatin factors functional in exit from pluripotency and the choice of proper lineages. Therefore, we quantitatively analyzed histone modifications in mouse ESC differentiation towards neuroectoderm and endoderm. Methods We cultured mouse ESCs (mESCs) and differentiated them towards neuroectoderm or endoderm lineages and performed mass spectrometry on total histones. Subsequent Western blots verified significantly altered H3K36me2. RT-qPCR analyses showed changes in H3K36-specific methyltransferases, demethylases and readers at mESC stage or during neuroectoderm/endoderm commitment. Results We presented quantitative histone modification levels in mESCs and lineage-committed cells. H3K36me2 increased specifically in neuroectoderm compared to mESCs or endoderm-committed cells. Regulation of H3K36 methylation might have a role in pluripotency exit and/or differentiation. Nsd2, Dnmt3b and Zmynd11 increased during differentiation regardless of lineage. Conversely, mESCs had higher Kdm4c and Msh6 expression than differentiated cells. Comparing neuroectoderm and endoderm-committed cells, we revealed Nsd1, Setd5 and Dnmt3a had lineage specific expression pattern. Conclusions Our results show quantitative changes in histone modifications during mESC lineage commitment and implicate H3K36me2 regulation for not only pluripotency exit but also lineage choice. Its regulatory proteins show stage (mESC vs. committed) or lineage (neuroectoderm vs. endoderm) dependent expression changes. Further work will be needed to discover their possible involvement in cell fate decisions and target genes.

Genome-wide screening in pluripotent cells identifies Mtf1 as a suppressor of mutant huntingtin toxicity

Huntington’s disease (HD) is a neurodegenerative disorder caused by CAG-repeat expansions in the huntingtin (HTT) gene. The resulting mutant HTT (mHTT) protein induces toxicity and cell death via multiple mechanisms and no effective therapy is available. Here, we employ a genome-wide screening in pluripotent mouse embryonic stem cells (ESCs) to identify suppressors of mHTT toxicity. Among the identified suppressors, linked to HD-associated processes, we focus on Metal response element binding transcription factor 1 (Mtf1). Forced expression of Mtf1 counteracts cell death and oxidative stress caused by mHTT in mouse ESCs and in human neuronal precursor cells. In zebrafish, Mtf1 reduces malformations and apoptosis induced by mHTT. In R6/2 mice, Mtf1 ablates motor defects and reduces mHTT aggregates and oxidative stress. Our screening strategy enables a quick in vitro identification of promising suppressor genes and their validation in vivo, and it can be applied to other monogenic diseases.

Semi-Supervised Non-Parametric Bayesian Modelling of Spatial Proteomics.

Understanding sub-cellular protein localisation is an essential component in the analysis of context specific protein function. Recent advances in quantitative mass-spectrometry (MS) have led to high resolution mapping of thousands of proteins to sub-cellular locations within the cell. Novel modelling considerations to capture the complex nature of these data are thus necessary. We approach analysis of spatial proteomics data in a non-parametric Bayesian framework, using K-component mixtures of Gaussian process regression models. The Gaussian process regression model accounts for correlation structure within a sub-cellular niche, with each mixture component capturing the distinct correlation structure observed within each niche. The availability of marker proteins (i.e. proteins with a priori known labelled locations) motivates a semi-supervised learning approach to inform the Gaussian process hyperparameters. We moreover provide an efficient Hamiltonian-within-Gibbs sampler for our model. Furthermore, we reduce the computational burden associated with inversion of covariance matrices by exploiting the structure in the covariance matrix. A tensor decomposition of our covariance matrices allows extended Trench and Durbin algorithms to be applied to reduce the computational complexity of inversion and hence accelerate computation. We provide detailed case-studies on Drosophila embryos and mouse pluripotent embryonic stem cells to illustrate the benefit of semi-supervised functional Bayesian modelling of the data.

Leucine and Arginine Availability Modulate Mouse Embryonic Stem Cell Proliferation and Metabolism.

Amino acids are crucial nutrients involved in several cellular and physiological processes, including fertilization and early embryo development. In particular, Leucine and Arginine have been shown to stimulate implantation, as lack of both in a blastocyst culture system is able to induce a dormant state in embryos. The aim of this work was to evaluate the effects of Leucine and Arginine withdrawal on pluripotent mouse embryonic stem cell status, notably, their growth, self-renewal, as well as glycolytic and oxidative metabolism. Our results show that the absence of both Leucine and Arginine does not affect mouse embryonic stem cell pluripotency, while reducing cell proliferation through cell-cycle arrest. Importantly, these effects are not related to Leukemia Inhibitory Factor (LIF) and are reversible when both amino acids are reconstituted in the culture media. Moreover, a lack of these amino acids is related to a reduction in glycolytic and oxidative metabolism and decreased protein translation in mouse embryonic stem cells (mESCs), while maintaining their pluripotent status.

Prdm14 promotes mouse ESC self-renewal and PGCLC specification through enhancement of Stat3 activity

SummaryPrdm14 plays an important role in the maintenance of mouse embryonic stem cell (mESC) pluripotency and the specification of primordial germ cells (PGCs). However, the mechanism downstream of Prdm14 is still not fully understood. Here, using high-throughput sequencing, chromatin immunoprecipitation, and luciferase reporter assays, we show that Prdm14 directly binds to the promoter of Socs3 and represses its transcription to increase the phosphorylation level of Stat3 protein, a critical downstream effector of LIF. Therefore, ectopic expression of Socs3 is able to decrease the ability of Prdm14 to promote mouse mESC self-renewal and PGC-like cell generation. As expected, similar phenotypes were observed in Prdm14-transfected mESCs after knockdown of Stat3 transcripts or treatment with a pan-inhibitor of JAKs, positive modulators of the LIF/Stat3 signaling pathway. These data will facilitate a better understanding of the regulatory network governing ESC identity and germ cell development.

OCT4 activates a Suv39h1-repressive antisense lncRNA to couple histone H3 Lysine 9 methylation to pluripotency.

Histone H3 Lysine 9 (H3K9) methylation, a characteristic mark of heterochromatin, is progressively implemented during development to contribute to cell fate restriction as differentiation proceeds. Accordingly, in undifferentiated and pluripotent mouse Embryonic Stem (ES) cells the global levels of H3K9 methylation are rather low and increase only upon differentiation. How global H3K9 methylation levels are coupled with the loss of pluripotency remains largely unknown. Here, we identify SUV39H1, a major H3K9 di- and tri-methylase, as an indirect target of the pluripotency network of Transcription Factors (TFs). We find that pluripotency TFs, principally OCT4, activate the expression of Suv39h1as, an antisense long non-coding RNA to Suv39h1. In turn, Suv39h1as downregulates Suv39h1 transcription in cis via a mechanism involving the modulation of the chromatin status of the locus. The targeted deletion of the Suv39h1as promoter region triggers increased SUV39H1 expression and H3K9me2 and H3K9me3 levels, affecting all heterochromatic regions, particularly peri-centromeric major satellites and retrotransposons. This increase in heterochromatinization efficiency leads to accelerated and more efficient commitment into differentiation. We report, therefore, a simple genetic circuitry coupling the genetic control of pluripotency with the global efficiency of H3K9 methylation associated with a major cell fate restriction, the irreversible loss of pluripotency.

Flow Cytometric Characterization of Pluripotent Cell Protein Markers in Naïve, Formative, and Primed Pluripotent Stem Cells.

Here we describe methodologies to characterize, delineate, and quantify pluripotent cells between naïve, formative, and primed pluripotent state mouse embryonic stem cell (mESCs) populations using flow cytometric analysis. This methodology can validate pluripotent states, sort individual cells of interest, and determine the efficiency of transitioning naïve mESCs to a primed-like state as mouse epiblast-like cells (mEpiLCs) and onto fully primed mouse epiblast stem cells (mEpiSCs). Quantification of the cell surface markers; SSEA1(CD15) and CD24 introduces an effective method of distinguishing individual cells from a population by their respective positioning in the pluripotent spectrum. Additionally, this protocol can be used to demarcate and sort cells via fluorescently activated cell sorting for downstream applications. Flow cytometric analysis within mESCs, mEpiLCs, and mEpiSCs can be efficiently completed using these optimized protocols.

MiR-290 family maintains developmental potential by targeting p21 in mouse preimplantation embryos‡.

The miR-290 family is a mouse-specific microRNA cluster, which maintains mouse embryonic stem cells (ESCs) pluripotency by increasing OCT3/4 and C-MYC expression. However, its functions in mouse preimplantation embryos remain unclear, especially during zygotic genome activation (ZGA). In this study, miR-290 family expression increased from the two-cell embryo stage through the blastocyst stage. Inhibition of miR-294-3p/5p did not affect ZGA initiation or embryo development, whereas pri-miR-290 knockdown decreased ZGA gene expression and slowed embryonic development. In addition, pluripotency decreased in ESCs derived from pri-miR-290 knockdown blastocysts. To clarify the mechanism of action, 33 candidate miR-294-3p target genes were screened from three databases, and miR-294-3p directly targeted the 3'-untranslated region of Cdkn1a (p21) mRNA. Similar to pri-miR-290 knockdown, P21 overexpression impeded embryonic development, whereas simultaneous overexpression of P21 and pri-miR-290 partially rescued embryonic development. The results indicate that the miR-290 family participates in promoting ZGA process and maintaining developmental potency in embryos by targeting p21.

A conserved expression signature predicts growth rate and reveals cell & lineage-specific differences.

Isogenic cells cultured together show heterogeneity in their proliferation rate. To determine the differences between fast and slow-proliferating cells, we developed a method to sort cells by proliferation rate, and performed RNA-seq on slow and fast proliferating subpopulations of pluripotent mouse embryonic stem cells (mESCs) and mouse fibroblasts. We found that slowly proliferating mESCs have a more naïve pluripotent character. We identified an evolutionarily conserved proliferation-correlated transcriptomic signature that is common to all eukaryotes: fast cells have higher expression of genes for protein synthesis and protein degradation. This signature accurately predicted growth rate in yeast and cancer cells, and identified lineage-specific proliferation dynamics during development, using C. elegans scRNA-seq data. In contrast, sorting by mitochondria membrane potential revealed a highly cell-type specific mitochondria-state related transcriptome. mESCs with hyperpolarized mitochondria are fast proliferating, while the opposite is true for fibroblasts. The mitochondrial electron transport chain inhibitor antimycin affected slow and fast subpopulations differently. While a major transcriptional-signature associated with cell-to-cell heterogeneity in proliferation is conserved, the metabolic and energetic dependency of cell proliferation is cell-type specific.

Inhibition of ubiquitin-specific protease 13-mediated degradation of Raf1 kinase by Spautin-1 has opposing effects in naïve and primed pluripotent stem cells

Embryonic stem cells (ESCs) are progenitor cells that retain the ability to differentiate into various cell types and are necessary for tissue repair. Improving cell culture conditions to maintain the pluripotency of ESCs in vitro is an urgent problem in the field of regenerative medicine. Here, we reveal that Spautin-1, a specific small-molecule inhibitor of ubiquitin-specific protease (USP) family members USP10 and USP13, promotes the maintenance of self-renewal and pluripotency of mouse ESCs in vitro. Functional studies reveal that only knockdown of USP13, but not USP10, is capable of mimicking the function of Spautin-1. Mechanistically, we demonstrate that USP13 physically interacts with, deubiquitinates, and stabilizes serine/threonine kinase Raf1 and thereby sustains Raf1 protein at the posttranslational level to activate the FGF/MEK/ERK prodifferentiation signaling pathway in naïve mouse ESCs. In contrast, in primed mouse epiblast stem cells and human induced pluripotent stem cells, the addition of Spautin-1 had an inhibitory effect on Raf1 levels, but USP13 overexpression promoted self-renewal. The addition of an MEK inhibitor impaired the effect of USP13 upregulation in these cells. These findings provide new insights into the regulatory network of naïve and primed pluripotency.

Site-specific O-GlcNAcylation of Psme3 maintains mouse stem cell pluripotency by impairing P-body homeostasis.

Mouse embryonic stem cell (ESC) pluripotency is tightly regulated by a complex network composed of extrinsic and intrinsic factors that allow proper organismal development. O-linked β-N-acetylglucosamine (O-GlcNAc) is the sole glycosylation mark found on cytoplasmic and nuclear proteins and plays a pivotal role in regulating fundamental cellular processes; however, its function in ESC pluripotency is still largely unexplored. Here, we identify O-GlcNAcylation of proteasome activator subunit 3 (Psme3) protein as a node of the ESC pluripotency network. Mechanistically, O-GlcNAc modification of serine 111 (S111) of Psme3 promotes degradation of Ddx6, which is essential for processing body (P-body) assembly, resulting in the maintenance of ESC pluripotent state. Conversely, loss of Psme3 S111 O-GlcNAcylation stabilizes Ddx6 and increases P-body levels, culminating in spontaneous exit of ESC from the pluripotent state. Our findings establish O-GlcNAcylation at S111 of Psme3 as a switch that regulates ESC pluripotency via control of P-body homeostasis.

LIF maintains mouse embryonic stem cells pluripotency by modulating TET1 and JMJD2 activity in a JAK2-dependent manner.

The LIF-JAK2-STAT3 pathway is the central signal transducer that maintains undifferentiated mouse embryonic stem cells (mESCs), which is achieved by the recruitment of activated STAT3 to the master pluripotency genes and activation of the gene transcriptions. It remains unclear, however, how the epigenetic status required for the master gene transcriptions is built into LIF-treated mESC cultures. In this study, Jak2, but not Stat3, in the LIF canonical pathway, establishes an open epigenetic status in the pluripotency gene promoter regions. Upon LIF activation, cytosolic JAK2 was translocalized into the nucleus of mESCs, and reduced DNA methylation (5mC levels) along with increasing DNA hydroxymethylation (5hmC) in the pluripotent gene (Nanog/Pou5f1) promoter regions. In addition, the repressive histone codes H3K9m3/H3K27m3 were reduced by JAK2. Activated JAK2 directly interacted with the core epigenetic enzymes TET1 and JMJD2, modulating its activity and promotes the DNA and histone demethylation, respectively. The JAK2 effects were attained by tyrosine phosphorylation on the epigenetic enzymes. The effects of JAK2 phosphorylation on the enzymes were diverse, but all were merged to the epigenetic signatures associated with open DNA/chromatin structures. Taken together, these results reveal a previously unrecognized epigenetic regulatory role of JAK2 as an important mediator of mESC maintenance.