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Here, we report a single-cell DNA replication sequencing method, scRepli-seq, a genome-wide methodology that measures copy number differences between replicated and unreplicated DNA. Using scRepli-seq, we demonstrate that replication-domain organization is conserved among individual mouse embryonic stem cells (mESCs). Differentiated mESCs exhibited distinct profiles, which were also conserved among cells. Haplotype-resolved scRepli-seq revealed similar replication profiles of homologous autosomes, while the inactive X chromosome was clearly replicated later than its active counterpart. However, a small degree of cell-to-cell replication-timing heterogeneity was present, which was smallest at the beginning and the end of S phase. In addition, developmentally regulated domains were found to deviate from others and showed a higher degree of heterogeneity, thus suggesting a link to developmental plasticity. Moreover, allelic expression imbalance was found to strongly associate with replication-timing asynchrony. Our results form a foundation for single-cell-level understanding of DNA replication regulation and provide insights into three-dimensional genome organization.

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs), which is a highly heterogeneous process. Here we report the cell fate continuum during somatic cell reprogramming at single-cell resolution. We first develop SOT to analyze cell fate continuum from Oct4/Sox2/Klf4- or OSK-mediated reprogramming and show that cells bifurcate into two categories, reprogramming potential (RP) or non-reprogramming (NR). We further show that Klf4 contributes to Cd34+/Fxyd5+/Psca+ keratinocyte-like NR fate and that IFN-γ impedes the final transition to chimera-competent pluripotency along the RP cells. We analyze more than 150,000 single cells from both OSK and chemical reprograming and identify additional NR/RP bifurcation points. Our work reveals a generic bifurcation model for cell fate decisions during somatic cell reprogramming that may be applicable to other systems and inspire further improvements for reprogramming., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Stem cell fate decisions are driven by a broad array of signals, both chemical and mechanical. Although much progress has been made in our understanding of the impact of chemical signals on cell fate choice, much less is known about the role and influence of mechanical signalling, particularly in embryonic stem (ES) cells. Many studies use substrates with different stiffness to study mechanical signalling, but changing substrate stiffness can induce secondary effects which are difficult to disentangle from the direct effects of forces/mechanical signals. To probe the direct impact of mechanical stress on cells, we developed an adaptable cell substrate stretcher to exert specific, reproducible forces on cells. Using this device to test the response of ES cells to tensile strain, we found that cells experienced a transient influx of calcium followed by an upregulation of the so-called immediate and early genes. On longer time scales, however, ES cells in ground state conditions were largely insensitive to mechanical stress. Nonetheless, as ES cells exited the ground state, their susceptibility to mechanical signals increased, resulting in broad transcriptional changes. Our findings suggest that exit from ground state of pluripotency is unaffected by mechanical signals, but that these signals could become important during the next stage of lineage specification. A better understanding of this process could improve our understanding of cell fate choice in early development and improve protocols for differentiation guided by mechanical cues.

Metazoan chromosomes are sequentially partitioned into topologically associating domains (TADs) and then into smaller sub-domains. One class of sub-domains, insulated neighborhoods, are proposed to spatially sequester and insulate the enclosed genes through self-association and chromatin looping. However, it has not been determined functionally whether promoter-enhancer interactions and gene regulation are broadly restricted to within these loops. Here, we employed published datasets from murine embryonic stem cells (mESCs) to identify insulated neighborhoods that confine promoter-enhancer interactions and demarcate gene regulatory regions. To directly address the functionality of these regions, we depleted estrogen-related receptor β (Esrrb), which binds the Mediator co-activator complex, to impair enhancers of genes within 222 insulated neighborhoods without causing mESC differentiation. Esrrb depletion reduces Mediator binding, promoter-enhancer looping, and expression of both nascent RNA and mRNA within the insulated neighborhoods without significantly affecting the flanking genes. Our data indicate that insulated neighborhoods represent functional regulons in mammalian genomes., (Copyright © 2018 Elsevier Inc. All rights reserved.)

During differentiation and reprogramming, new cell identities are generated by reconfiguration of gene regulatory networks. Here, we combined automated formal reasoning with experimentation to expose the logic of network activation during induction of naïve pluripotency. We find that a Boolean network architecture defined for maintenance of naïve state embryonic stem cells (ESC) also explains transcription factor behaviour and potency during resetting from primed pluripotency. Computationally identified gene activation trajectories were experimentally substantiated at single-cell resolution by RT-qPCR Contingency of factor availability explains the counterintuitive observation that Klf2, which is dispensable for ESC maintenance, is required during resetting. We tested 124 predictions formulated by the dynamic network, finding a predictive accuracy of 77.4%. Finally, we show that this network explains and predicts experimental observations of somatic cell reprogramming. We conclude that a common deterministic program of gene regulation is sufficient to govern maintenance and induction of naïve pluripotency. The tools exemplified here could be broadly applied to delineate dynamic networks underlying cell fate transitions., (© 2018 The Authors. Published under the terms of the CC BY 4.0 license.)

The differentiation of mouse embryonic stem (ES) cells into skeletal myocytes can be enhanced by small molecular inducers, which signal through muscle-specific transcription networks. However, to induce differentiation in vitro, the ES cells must be thrusted through the stage of embryoid body (EB) formation. Here, we describe how to efficiently direct the commitment of ES cells into skeletal muscle lineage by using nuclear receptor agonists in a hanging drop protocol.

The commitment of pluripotent stem cells to the cardiac lineage has enormous potential in regenerative medicine interventions for several cardiac diseases. Thus, it is necessary to understand and regulate this differentiation process for potential clinical application. In this study, we developed defined conditions with chemical inducers for effective cardiac lineage commitment and elucidated the mechanism for high-efficiency differentiation. First, we designed a robust reporter-based platform to screen chemical inducers of cardiac differentiation in the mouse P19 teratocarcinoma cell line. Using this system, we identified two natural alkaloids, lupinine and ursinoic acid, which enhanced cardiomyocyte differentiation of P19 cells in terms of beating colony numbers with respect to oxytocin, and confirmed their activity in mouse embryonic stem cells. By analyzing the expression of key markers, we found that this enhancement can be attributed to the early and rapid induction of the Wnt signaling pathway. We also found that these natural compounds could not only supersede the action of the Wnt3a ligand but also had a very quick response time, allowing them to act as efficient cardiac mesoderm inducers that subsequently promoted cardiomyocyte differentiation. Thus, this study offers a way to develop chemical-based differentiation strategy for high-efficiency cardiac lineage commitment, which has an advantage over currently available methods with complex medium composition and parameters. Furthermore, it also provides an opportunity to pinpoint the key molecular mechanisms pivotal to the cardiac differentiation process, which are necessary to design an efficient strategy for cardiomyocyte differentiation.

Profiling the transcriptomes of individual cells with single-cell RNA sequencing (scRNA-seq) has been widely applied to provide a detailed molecular characterization of cellular heterogeneity within a population of cells. Despite recent technological advances of scRNA-seq, technical variability of gene expression in scRNA-seq is still much higher than that in bulk RNA-seq. Accounting for technical variability is therefore a prerequisite for correctly analyzing single-cell data. This chapter describes a computational pipeline for detecting highly variable genes exhibiting higher cell-to-cell variability than expected by technical noise. The basic pipeline using the scater and scran R/Bioconductor packages includes deconvolution-based normalization, fitting the mean-variance trend, testing for nonzero biological variability, and visualization with highly variable genes. An outline of the underlying theory of detecting highly variable genes is also presented. We illustrate how the pipeline works by using two case studies, one from mouse embryonic stem cells with external RNA spike-ins, and the other from mouse dentate gyrus cells without spike-ins.

Genome editing occurs in the context of chromatin, which is heterogeneous in structure and function across the genome. Chromatin heterogeneity is thought to affect genome editing efficiency, but this has been challenging to quantify due to the presence of confounding variables. Here, we develop a method that exploits the allele-specific chromatin status of imprinted genes in order to address this problem in cycling mouse embryonic stem cells (mESCs). Because maternal and paternal alleles of imprinted genes have identical DNA sequence and are situated in the same nucleus, allele-specific differences in the frequency and spectrum of mutations induced by CRISPR-Cas9 can be unequivocally attributed to epigenetic mechanisms. We found that heterochromatin can impede mutagenesis, but to a degree that depends on other key experimental parameters. Mutagenesis was impeded by up to 7-fold when Cas9 exposure was brief and when intracellular Cas9 expression was low. In contrast, the outcome of mutagenic DNA repair was unaffected by chromatin state, with similar efficiencies of homology-directed repair (HDR) and deletion spectra on maternal and paternal chromosomes. Combined, our data show that heterochromatin imposes a permeable barrier that influences the kinetics, but not the endpoint, of CRISPR-Cas9 genome editing and suggest that therapeutic applications involving low-level Cas9 exposure will be particularly affected by chromatin status., Competing Interests: The authors have declared that no competing interests exist.

The genome of pluripotent stem cells adopts a unique three-dimensional architecture featuring weakly condensed heterochromatin and large nucleosome-free regions. Yet, it is unknown whether structural loops and contact domains display characteristics that distinguish embryonic stem cells (ESCs) from differentiated cell types. We used genome-wide chromosome conformation capture and super-resolution imaging to determine nuclear organization in mouse ESC and neural stem cell (NSC) derivatives. We found that loss of pluripotency is accompanied by widespread gain of structural loops. This general architectural change correlates with enhanced binding of CTCF and cohesins and more pronounced insulation of contacts across chromatin boundaries in lineage-committed cells. Reprogramming NSCs to pluripotency restores the unique features of ESC domain topology. Domains defined by the anchors of loops established upon differentiation are enriched for developmental genes. Chromatin loop formation is a pervasive structural alteration to the genome that accompanies exit from pluripotency and delineates the spatial segregation of developmentally regulated genes., (Published by Elsevier Inc.)

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome editing, embryonic stem (ES) cell-based gene-targeting technology does not have these shortcomings and is widely used to modify animal/mouse genome ranging from large deletions/insertions to single nucleotide substitutions. Notably, identifying the relatively few homologous recombination (HR) events necessary to obtain desired ES clones is a key step, which demands accurate and reliable methods. Southern blotting and/or conventional PCR are often utilized for this purpose. Here, we describe the detailed procedures of using those two methods to identify HR events that occurred in mouse ES cells in which the endogenous Myh9 gene is intended to be disrupted and replaced by cDNAs encoding other nonmuscle myosin heavy chain IIs (NMHC IIs). The whole procedure of Southern blotting includes the construction of targeting vector(s), electroporation, drug selection, the expansion and storage of ES cells/clones, the preparation, digestion, and blotting of genomic DNA (gDNA), the hybridization and washing of probe(s), and a final step of autoradiography on the X-ray films. PCR can be performed directly with prepared and diluted gDNA. To obtain ideal results, the probes and restriction enzyme (RE) cutting sites for Southern blotting and the primers for PCR should be carefully planned. Though the execution of Southern blotting is time-consuming and labor-intensive and PCR results have false positives, the correct identification by Southern blotting and the rapid screening by PCR allow the sole or combined application of these methods described in this paper to be widely used and consulted by most labs in the identification of genotypes of ES cells and genetically modified animals.

Objective: Despite a great number of studies analysing the effects of microgravity on stem cell proliferation and differentiation, few of them have focused on real-time imaging estimates in space. Herein, we utilized the TZ-1 cargo spacecraft, automatic cell culture equipment and live cell imaging techniques to examine the effects of real microgravity on the proliferation and differentiation of mouse embryonic stem cells (mESCs)., Materials and Methods: Oct4-GFP, Brachyury-GFP mESC and Oct4-GFP mESC-derived EBs were used as experimental samples in the TZ-1 spaceflight mission. These samples were seeded into chambers, cultured in an automatic cell culture device and were transported into space during the TZ-1 mission. Over 15 days of spaceflight, bright field and fluorescent images of cell growth were taken in micrography, and the medium was changed every day. Real-time image data were transferred to the ground for analysis., Results: Space microgravity maintains stemness and long-term survival of mESCs, promising 3D aggregate formation. Although microgravity did not significantly prevent the migration of EBs on the ECM substrate, it did prevent terminal differentiation of cells., Conclusions: This study demonstrates that space microgravity might play a potential role in supporting 3D cell growth and maintenance of stemness in embryonic stem cells, while it may negatively affect terminal differentiation., (© 2018 John Wiley & Sons Ltd.)

In embryonic stem cells (ESCs), developmental gene promoters are characterized by their bivalent chromatin state, with simultaneous modification by MLL2 and Polycomb complexes. Although essential for embryogenesis, bivalency is functionally not well understood. Here, we show that MLL2 plays a central role in ESC genome organization. We generate a catalog of bona fide bivalent genes in ESCs and demonstrate that loss of MLL2 leads to increased Polycomb occupancy. Consequently, promoters lose accessibility, long-range interactions are redistributed, and ESCs fail to differentiate. We pose that bivalency balances accessibility and long-range connectivity of promoters, allowing developmental gene expression to be properly modulated.

Background: Whether the adult mammalian heart harbors cardiac stem cells for regeneration of cardiomyocytes is an important yet contentious topic in the field of cardiovascular regeneration. The putative myocyte stem cell populations recognized without specific cell markers, such as the cardiosphere-derived cells, or with markers such as Sca1 + , Bmi1 + , Isl1 + , or Abcg2 + cardiac stem cells have been reported. Moreover, it remains unclear whether putative cardiac stem cells with unknown or unidentified markers exist and give rise to de novo cardiomyocytes in the adult heart., Methods: To address this question without relying on a particular stem cell marker, we developed a new genetic lineage tracing system to label all nonmyocyte populations that contain putative cardiac stem cells. Using dual lineage tracing system, we assessed whether nonmyocytes generated any new myocytes during embryonic development, during adult homeostasis, and after myocardial infarction. Skeletal muscle was also examined after injury for internal control of new myocyte generation from nonmyocytes., Results: By this stem cell marker-free and dual recombinases-mediated cell tracking approach, our fate mapping data show that new myocytes arise from nonmyocytes in the embryonic heart, but not in the adult heart during homeostasis or after myocardial infarction. As positive control, our lineage tracing system detected new myocytes derived from nonmyocytes in the skeletal muscle after injury., Conclusions: This study provides in vivo genetic evidence for nonmyocyte to myocyte conversion in embryonic but not adult heart, arguing again the myogenic potential of putative stem cell populations for cardiac regeneration in the adult stage. This study also provides a new genetic strategy to identify endogenous stem cells, if any, in other organ systems for tissue repair and regeneration.

Embryonic stem cells can be incorporated into the developing embryo and its germ line, but, when cultured alone, their ability to generate embryonic structures is restricted. They can interact with trophoblast stem cells to generate structures that break symmetry and specify mesoderm, but their development is limited as the epithelial-mesenchymal transition of gastrulation cannot occur. Here, we describe a system that allows assembly of mouse embryonic, trophoblast and extra-embryonic endoderm stem cells into structures that acquire the embryo's architecture with all distinct embryonic and extra-embryonic compartments. Strikingly, such embryo-like structures develop to undertake the epithelial-mesenchymal transition, leading to mesoderm and then definitive endoderm specification. Spatial transcriptomic analyses demonstrate that these morphological transformations are underpinned by gene expression patterns characteristic of gastrulating embryos. This demonstrates the remarkable ability of three stem cell types to self-assemble in vitro into gastrulating embryo-like structures undertaking spatio-temporal events of the gastrulating mammalian embryo.

The emergence of form and function during mammalian embryogenesis is a complex process that involves multiple regulatory levels. The foundations of the body plan are laid throughout the first days of post-implantation development as embryonic stem cells undergo symmetry breaking and initiate lineage specification, in a process that coincides with a global morphological reorganization of the embryo. Here, we review experimental models and how they have shaped our current understanding of the post-implantation mammalian embryo.

Pluripotency is accompanied by the erasure of parental epigenetic memory, with naïve pluripotent cells exhibiting global DNA hypomethylation both in vitro and in vivo. Exit from pluripotency and priming for differentiation into somatic lineages is associated with genome-wide de novo DNA methylation. We show that during this phase, co-expression of enzymes required for DNA methylation turnover, DNMT3s and TETs, promotes cell-to-cell variability in this epigenetic mark. Using a combination of single-cell sequencing and quantitative biophysical modeling, we show that this variability is associated with coherent, genome-scale oscillations in DNA methylation with an amplitude dependent on CpG density. Analysis of parallel single-cell transcriptional and epigenetic profiling provides evidence for oscillatory dynamics both in vitro and in vivo. These observations provide insights into the emergence of epigenetic heterogeneity during early embryo development, indicating that dynamic changes in DNA methylation might influence early cell fate decisions., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

A large body of data have accumulated that characterize the gene regulatory network of stem cells. Yet, a comprehensive and integrative understanding of this complex network is lacking. Network reverse engineering methods that use transcriptome data to derive these networks may help to uncover the topology in an unbiased way. Many methods exist that use co-expression to reconstruct networks. However, it remains unclear how these methods perform in the context of stem cell differentiation, as most systematic assessments have been made for regulatory networks of unicellular organisms. Here, we report a systematic benchmark of different reverse engineering methods against functional data. We show that network pruning is critical for reconstruction performance. We also find that performance is similar for algorithms that use different co-expression measures, i.e. mutual information or correlation. In addition, different methods yield very different network topologies, highlighting the challenge of interpreting these resulting networks as a whole.This article is part of the theme issue 'Designer human tissue: coming to a lab near you'., (© 2018 The Author(s).)

Abstact: Polycomb-mediated repression of gene expression is essential for development, with a pivotal role played by trimethylation of histone H3 lysine 27 (H3K27me3), which is deposited by Polycomb Repressive Complex 2 (PRC2). The mechanism by which PRC2 is recruited to target genes has remained largely elusive, particularly in vertebrates. Here we demonstrate that MTF2, one of the three vertebrate homologs of Drosophila melanogaster Polycomblike, is a DNA-binding, methylation-sensitive PRC2 recruiter in mouse embryonic stem cells. MTF2 directly binds to DNA and is essential for recruitment of PRC2 both in vitro and in vivo. Genome-wide recruitment of the PRC2 catalytic subunit EZH2 is abrogated in Mtf2 knockout cells, resulting in greatly reduced H3K27me3 deposition. MTF2 selectively binds regions with a high density of unmethylated CpGs in a context of reduced helix twist, which distinguishes target from non-target CpG islands. These results demonstrate instructive recruitment of PRC2 to genomic targets by MTF2.

STAT3 phosphorylation at tyrosine 705 (STAT3 pY705 ), triggered by the addition of the leukemia inhibitory factor (LIF), can maintain mouse embryonic stem cell (mESC) self-renewal and reprogram mouse epiblast stem cells (EpiSCs) to enter a naïve pluripotent state. The activation of STAT3 pY705 occurs mainly through Janus kinases. However, it remains unclear how STAT3 pY705 levels are decreased in mESCs. Our study shows that upregulation of the protein tyrosine phosphatase (PTPN2) inhibits STAT3 activity by reducing its phosphorylation level and promotes mESC differentiation, whereas PTPN2 knockout by CRISPR/CAS9 delays mESC differentiation. Consistently, PTPN2 knockdown facilitates the generation of mESC-like colonies in STAT3-overexpressing EpiSCs. PTPN2-mediated STAT3 activity, thus, contributes to the exit of ESCs from the pluripotent ground state. These findings expand the current understanding of the regulatory network of naïve pluripotency., (© 2018 Federation of European Biochemical Societies.)

Mammalian embryogenesis requires the coordination of embryonic and extra-embryonic tissues to enable implantation into the uterus and post-implantation development to establish the body plan. Mouse embryonic stem cells (ESCs) are a useful tool for studying pluripotent embryonic tissue in vitro. However, they cannot undertake correct embryogenesis alone. Many attempts to model the early embryo in vitro involve the aggregation of ESCs into spheroids of variable size and cell number that undertake germ-layer specification but fail to recapitulate the characteristic architecture and arrangement of tissues of the early embryo. Here, we describe a protocol to generate the first embryo-like structures by directing the assembly of mouse ESCs and extra-embryonic trophoblast stem cells (TSCs) in a 3D extracellular matrix (ECM) into structures we call 'polarized embryo-like structures'. By establishing the medium and culture conditions needed to support the growth of both stem cell types simultaneously, embryonic architecture is generated within 4 d of co-culture. This protocol can be performed by those proficient in standard ESC culture techniques and can be used in developmental studies to investigate the interactions between embryonic and extra-embryonic tissues during mammalian development.

Haploid mouse embryonic stem cells (ESCs), in which a single hit mutation is sufficient to produce loss-of-function phenotypes, have provided a powerful tool for forward genetic screening. This strategy, however, can be hampered by undesired autodiploidization of haploid ESCs. To overcome this obstacle, we designed a new methodology that facilitates enrichment of homozygous mutant ESC clones arising from autodiploidization during haploid gene trap mutagenesis. Haploid mouse ESCs were purified by fluorescence-activated cell sorting to maintain their haploid property and then transfected with the Tol2 transposon-based biallelically polyA-trapping (BPATrap) vector that carries an invertible G418 plus puromycin double selection cassette. G418 plus puromycin double selection enriched biallelic mutant clones that had undergone autodiploidization following a single vector insertion into the haploid genome. Using this method, we successfully generated 222 homozygous mutant ESCs from 2208 clones by excluding heterozygous ESCs and ESCs with multiple vector insertions. This relatively low efficiency of generating homozygous mutant ESCs was partially overcome by cell sorting of haploid ESCs after Tol2 BPATrap transfection. These results demonstrate the feasibility of our approach to provide an efficient platform for mutagenesis of ESCs and functional analysis of the mammalian genome.

The mammary gland is composed of basal cells and luminal cells. It is generally believed that the mammary gland arises from embryonic multipotent progenitors, but it remains unclear when lineage restriction occurs and what mechanisms are responsible for the switch from multipotency to unipotency during its morphogenesis. Here, we perform multicolour lineage tracing and assess the fate of single progenitors, and demonstrate the existence of a developmental switch from multipotency to unipotency during embryonic mammary gland development. Molecular profiling and single cell RNA-seq revealed that embryonic multipotent progenitors express a unique hybrid basal and luminal signature and the factors associated with the different lineages. Sustained p63 expression in embryonic multipotent progenitors promotes unipotent basal cell fate and was sufficient to reprogram adult luminal cells into basal cells by promoting an intermediate hybrid multipotent-like state. Altogether, this study identifies the timing and the mechanisms mediating early lineage segregation of multipotent progenitors during mammary gland development.

The metameric structure in vertebrates is based on the periodic formation of somites from the anterior end of the presomitic mesoderm (PSM). The segmentation boundary is defined by the Tbx6 expression domain, whose anterior limit is determined by Tbx6 protein destabilization via Ripply2. However, the molecular mechanism of this process is poorly understood. Here, we show that Ripply2 directly binds to Tbx6 in cultured cells without changing the stability of Tbx6, indicating an unknown mechanism for Tbx6 degradation in vivo. We succeeded in reproducing in vivo events using a mouse ES induction system, in which Tbx6 degradation occurred via Ripply2. Mass spectrometry analysis of the PSM-fated ES cells revealed that proteasomes are major components of the Ripply2-binding complex, suggesting that recruitment of a protein-degradation-complex is a pivotal function of Ripply2. Finally, we identified a motif in the T-box, which is required for Tbx6 degradation independent of binding with Ripply2 in vivo., Competing Interests: WZ, MO, RA, MK, AO, YS No competing interests declared, (© 2018, Zhao et al.)

This protocol is an extension to: Nat. Protoc. 6, 870-895 (2014); doi:10.1038/nprot.2011.328; published online 02 June 2011Cycling cells duplicate their DNA content during S phase, following a defined program called replication timing (RT). Early- and late-replicating regions differ in terms of mutation rates, transcriptional activity, chromatin marks and subnuclear position. Moreover, RT is regulated during development and is altered in diseases. Here, we describe E/L Repli-seq, an extension of our Repli-chip protocol. E/L Repli-seq is a rapid, robust and relatively inexpensive protocol for analyzing RT by next-generation sequencing (NGS), allowing genome-wide assessment of how cellular processes are linked to RT. Briefly, cells are pulse-labeled with BrdU, and early and late S-phase fractions are sorted by flow cytometry. Labeled nascent DNA is immunoprecipitated from both fractions and sequenced. Data processing leads to a single bedGraph file containing the ratio of nascent DNA from early versus late S-phase fractions. The results are comparable to those of Repli-chip, with the additional benefits of genome-wide sequence information and an increased dynamic range. We also provide computational pipelines for downstream analyses, for parsing phased genomes using single-nucleotide polymorphisms (SNPs) to analyze RT allelic asynchrony, and for direct comparison to Repli-chip data. This protocol can be performed in up to 3 d before sequencing, and requires basic cellular and molecular biology skills, as well as a basic understanding of Unix and R.

Mammalian haploid embryonic stem cells (haESCs) serve as a powerful tool for genetic analyses at both the cellular and organismal levels. However, spontaneous diploidization of haESCs limits their use in these analyses. Addition of small molecules to the culture medium to control the cell cycle can slow down diploidization, but cell-sorting methods such as FACS are still required to enrich haploid cells for long-term maintenance in vitro Here, acting on our observation that haploid and diploidized cells differ in diameter, we developed a simplified filtration method to enrich haploid cells from cultured haESCs. We found that regular cell filtration with this system reliably maintained the haploidy of mouse haESCs for over 30 passages. Importantly, CRISPR/Cas9-mediated knockout and knockin were successfully achieved in the filtered cells, leading to stable haploid cell lines carrying the desired gene modifications. Of note, by injecting haESCs into metaphase II oocytes, we efficiently obtained live mice with the expected genetic traits, indicating that regular filtration maintained the functional integrity of haESCs. Moreover, this filtration system was also feasible for derivation of mouse haESCs from parthenogenetic haploid blastocysts and for human haESC maintenance. In conclusion, we have identified a reliable, efficient, and easy-to-handle technique for countering diploidization of haploid cells, a major obstacle in haESC applications., (© 2018 Qu et al.)

The capability of generating neural precursor cells with distinct types of regional identity in vitro has recently opened new opportunities for cell replacement in animal models of neurodegenerative diseases. By manipulating Wnt and BMP signaling, we steered the differentiation of mouse embryonic stem cells (ESCs) toward isocortical or hippocampal molecular identity. These two types of cells showed different degrees of axonal outgrowth and targeted different regions when co-transplanted in healthy or lesioned isocortex or in hippocampus. In hippocampus, only precursor cells with hippocampal molecular identity were able to extend projections, contacting CA3. Conversely, isocortical-like cells were capable of extending long-range axonal projections only when transplanted in motor cortex, sending fibers toward both intra- and extra-cortical targets. Ischemic damage induced by photothrombosis greatly enhanced the capability of isocortical-like cells to extend far-reaching projections. Our results indicate that neural precursors generated by ESCs carry intrinsic signals specifying axonal extension in different environments., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

The transition from naive to primed state of pluripotent stem cells is hallmarked by epithelial-mesenchymal transition, metabolic switch from oxidative phosphorylation to aerobic glycolysis, and changes in the epigenetic landscape. Since these changes are also seen as putative hallmarks of neoplastic cell transformation, we hypothesized that oncogenic pathways may be involved in this process. We report that the activity of RAS is repressed in the naive state of mouse embryonic stem cells (ESCs) and that all three RAS isoforms are significantly activated upon early differentiation induced by LIF withdrawal, embryoid body formation, or transition to the primed state. Forced expression of active RAS and RAS inhibition have shown that RAS regulates glycolysis, CADHERIN expression, and the expression of repressive epigenetic marks in pluripotent stem cells. Altogether, this study indicates that RAS is located at a key junction of early ESC differentiation controlling key processes in priming of naive cells., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Stem cells are defined by their capacities to self-renew and generate progeny of multiple lineages. The transcription factor SOX2 has key roles in the regulation of stem cell characteristics, but whether SOX2 achieves these functions through similar mechanisms in distinct stem cell populations is not known. To address this question, we performed RNA-seq and SOX2 ChIP-seq on embryonic mouse cortex, spinal cord, stomach and lung/esophagus. We demonstrate that, although SOX2 binds a similar motif in the different cell types, its target regions are primarily cell-type-specific and enriched for the distinct binding motifs of appropriately expressed interacting co-factors. Furthermore, cell-type-specific SOX2 binding in endodermal and neural cells is most often found around genes specifically expressed in the corresponding tissue. Consistent with this, we demonstrate that SOX2 target regions can act as cis-regulatory modules capable of directing reporter expression to appropriate tissues in a zebrafish reporter assay. In contrast, SOX2 binding sites found in both endodermal and neural tissues are associated with genes regulating general stem cell features, such as proliferation. Notably, we provide evidence that SOX2 regulates proliferation through conserved mechanisms and target genes in both germ layers examined. Together, these findings demonstrate how SOX2 simultaneously regulates cell-type-specific, as well as core transcriptional programs in neural and endodermal stem cells.

Induced pluripotent stem cells can be derived from somatic cells through ectopic expression of transcription factors or chemical cocktails. Chemical iPSCs (C-iPSCs) and OSKM-iPSCs (4F-iPSCs) have been suggested to have similar characteristics to mouse embryonic stem cells (mESCs). However, their epigenetic equivalence remains incompletely understood throughout the genome. In this study, we have generated mouse C-iPSCs and 4F-iPSCs, and further compared the genome-wide DNA methylomes of C-iPSCs, 4F-iPSCs, and mESCs that were maintained in 2i and LIF. Three pluripotent stem cells tend to be low methylated overall, however, DNA methylations in some specific regions (such as retrotransposons) are cell type-specific. Importantly, C-iPSCs are more hypomethylated than 4F-iPSCs. Bisulfite sequencing indicated that DNA methylation status in several known imprinted clusters, such as: Dlk1-Dio3 and Peg12-Ube3a, in C-iPSCs are closer to those of mESCs than 4F-iPSCs. Overall, our data demonstrate the reprogramming methods-dependent epigenetic differences of C-iPSCs and 4F-iPSCs and reveal that C-iPSCs are more hypomethylated than OSKM-integrated iPSCs.

Diverse Polycomb repressive complexes 1 (PRC1) play essential roles in gene regulation, differentiation and development. Six major groups of PRC1 complexes that differ in their subunit composition have been identified in mammals. How the different PRC1 complexes are recruited to specific genomic sites is poorly understood. The Polycomb Ring finger protein PCGF6, the transcription factors MGA and E2F6, and the histone-binding protein L3MBTL2 are specific components of the non-canonical PRC1.6 complex. In this study, we have investigated their role in genomic targeting of PRC1.6. ChIP-seq analysis revealed colocalization of MGA, L3MBTL2, E2F6 and PCGF6 genome-wide. Ablation of MGA in a human cell line by CRISPR/Cas resulted in complete loss of PRC1.6 binding. Rescue experiments revealed that MGA recruits PRC1.6 to specific loci both by DNA binding-dependent and by DNA binding-independent mechanisms. Depletion of L3MBTL2 and E2F6 but not of PCGF6 resulted in differential, locus-specific loss of PRC1.6 binding illustrating that different subunits mediate PRC1.6 loading to distinct sets of promoters. Mga, L3mbtl2 and Pcgf6 colocalize also in mouse embryonic stem cells, where PRC1.6 has been linked to repression of germ cell-related genes. Our findings unveil strikingly different genomic recruitment mechanisms of the non-canonical PRC1.6 complex, which specify its cell type- and context-specific regulatory functions.

Background: Stem cells-derived neuronal cultures hold great promise for in vitro disease modelling and drug screening. However, currently stem cells-derived neuronal cultures do not recapitulate the functional properties of primary neurons, such as network properties. Cultured primary murine neurons develop networks which are synchronised over large fractions of the culture, whereas neurons derived from mouse embryonic stem cells (ESCs) display only partly synchronised network activity and human pluripotent stem cells-derived neurons have mostly asynchronous network properties. Therefore, strategies to improve correspondence of derived neuronal cultures with primary neurons need to be developed to validate the use of stem cell-derived neuronal cultures as in vitro models., New Method: By combining serum-free derivation of ESCs from mouse blastocysts with neuronal differentiation of ESCs in morphogen-free adherent culture we generated neuronal networks with properties recapitulating those of mature primary cortical cultures., Results: After 35days of differentiation ESC-derived neurons developed network activity very similar to that of mature primary cortical neurons. Importantly, ESC plating density was critical for network development., Comparison With Existing Method(s): Compared to the previously published methods this protocol generated more synchronous neuronal networks, with high similarity to the networks formed in mature primary cortical culture., Conclusion: We have demonstrated that ESC-derived neuronal networks recapitulating key properties of mature primary cortical networks can be generated by optimising both stem cell derivation and differentiation. This validates the approach of using ESC-derived neuronal cultures for disease modelling and in vitro drug screening., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Deletion of Sox2 from mouse embryonic stem cells (ESCs) causes trophectodermal differentiation. While this can be prevented by enforced expression of the related SOXB1 proteins, SOX1 or SOX3, the roles of SOXB1 proteins in epiblast stem cell (EpiSC) pluripotency are unknown. Here, we show that Sox2 can be deleted from EpiSCs with impunity. This is due to a shift in the balance of SoxB1 expression in EpiSCs, which have decreased Sox2 and increased Sox3 compared to ESCs. Consistent with functional redundancy, Sox3 can also be deleted from EpiSCs without eliminating self-renewal. However, deletion of both Sox2 and Sox3 prevents self-renewal. The overall SOXB1 levels in ESCs affect differentiation choices: neural differentiation of Sox2 heterozygous ESCs is compromised, while increased SOXB1 levels divert the ESC to EpiSC transition towards neural differentiation. Therefore, optimal SOXB1 levels are critical for each pluripotent state and for cell fate decisions during exit from naïve pluripotency.

The variability in embryonic stem (ES) cell culture is due primarily to serum. Serum is typically produced in large batches from many animals. However, samples may differ depending on the age and diet of the animals, the country of origin, and other factors creating lot-to-lot variations. Some vendors test FBS lots for compatibility with ES cell culture. Many laboratories prefer to test serum batches themselves to identify the lot giving optimal growth. In this protocol, small quantities of specific serum batches are obtained from different suppliers and tested for their ability to support ES cells in an undifferentiated state. A complete test includes the serum batches' influence on plating efficiency, cell morphology, toxicity, and, if possible, their ability to support generation of chimeras., (© 2017 Cold Spring Harbor Laboratory Press.)

Single-cell RNA sequencing (scRNA-seq) is a powerful method for dissecting intercellular heterogeneity during development. Conventional trajectory analysis provides only a pseudotime of development, and often discards cell-cycle events as confounding factors. Here using matched cell population RNA-seq (cpRNA-seq) as a reference, we developed an "iCpSc" package for integrative analysis of cpRNA-seq and scRNA-seq data. By generating a computational model for reference "biological differentiation time" using cell population data and applying it to single-cell data, we unbiasedly associated cell-cycle checkpoints to the internal molecular timer of single cells. Through inferring a network flow from cpRNA-seq to scRNA-seq data, we predicted a role of M phase in controlling the speed of neural differentiation of mouse embryonic stem cells, and validated it through gene knockout (KO) experiments. By linking temporally matched cpRNA-seq and scRNA-seq data, our approach provides an effective and unbiased approach for identifying developmental trajectory and timing-related regulatory events.

The ability to propagate mature cells and tissue from pluripotent stem cells offers enormous promise for treating many diseases, including neurodegenerative diseases. Before such cells can be used successfully in neurodegenerative diseases without causing unwanted cell growth and migration, genes regulating growth and migration of neural stem cells need to be well characterized. Estrogen receptor beta (ERβ) is essential for migration of neurons and glial cells in the developing mouse brain. To examine whether ERβ influences differentiation of mouse embryonic stem cells (mESC) into neural lineages, we compared control and ERβ knockout (BERKO) mESCs at defined stages of neural development and examined the effects of an ERβ-selective ligand (LY3201) with a combination of global and targeted gene-expression profiling and the expression of key pluripotency markers. We found that ERβ was induced in embryoid bodies (EBs) and neural precursor cells (NPCs) during development. Proliferation was higher in BERKO NPCs and was inhibited by LY3201. Neurogenesis was reduced in BERKO ES cells, and oligodendrogliogenesis was enhanced. BERKO EBs expressed higher levels of key ectodermal and neural progenitor markers and lower levels of markers for mesoderm and endoderm lineages. ERβ-regulated factors are involved in cell adhesion, axon guidance, and signaling of Notch and GABA receptor pathways, as well as factors important for the differentiation of neuronal precursors into dopaminergic neurons (Engrailed 1) and for the oligodendrocyte fate acquisition (Olig2). Our data suggest that ERβ is an important component for differentiation into midbrain neurons as well as for preventing precocious oligodendrogliogenesis., Competing Interests: The authors declare no conflict of interest.

TFCP2L1 is a transcription factor that is crucial for self-renewal of mouse embryonic stem cells (mESCs). How TFCP2L1 maintains the pluripotent state of mESCs, however, remains unknown. Here, we show that knockdown of Tfcp2l1 in mESCs induces the expression of endoderm, mesoderm and trophectoderm markers. Functional analysis of mutant forms of TFCP2L1 revealed that TFCP2L1 depends on its N-terminus and CP2-like domain to maintain the undifferentiated state of mESCs. The N-terminus of TFCP2L1 is mainly associated with the suppression of mesoderm and trophectoderm differentiation, while the CP2-like domain is closely related to the suppression of endoderm commitment. Further studies showed that MTA1 directly interacts with TFCP2L1 and is indispensable for the TFCP2L1-mediated self-renewal-promoting effect and endoderm-inhibiting action. TFCP2L1-mediated suppression of mesoderm and trophectoderm differentiation, however, seems to be due to downregulation of Lef1 expression. Our study thus provides an expanded understanding of the function of TFCP2L1 and the pluripotency regulation network of ESCs., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Background: Self-renewal and differentiation of embryonic stem cells (ESCs) is directed by biological and/or physical cues that regulate multiple signaling cascades. We have previously shown that mESCs seeded in a type I collagen matrix demonstrate a loss of pluripotent marker expression and differentiate towards an osteogenic lineage. In this study, we examined if this effect was mediated in part through Arginylglycylaspartic acid (RGD) dependent integrin activity and/or mechano-transduction., Results: The results from this study suggest that mESC interaction with the local microenvironment through RGD dependent integrins play a role in the regulation of mESC core transcription factors (TF), Oct-4, Sox 2 and Nanog. Disruption of this interaction with a cyclic RGD peptide (cRGDfC) was sufficient to mimic the effect of a mechanical stimulus in terms of pluripotent gene expression, specifically, we observed that supplementation with cRGDfC, or mechanical stimulus, significantly influenced mESC pluripotency by down-regulating core transcription factors. Moreover, our results indicated that the presence of the cRGDfC peptide inhibited integrin expression and up-regulated early lineage markers (mesoderm and ectoderm) in a Leukemia inhibitory factor (LIF) dependent manner. When cRGDfC treated mESCs were injected in Severe combined immunodeficiency (SCID) mice, no tissue growth and/or teratoma formation was observed, suggesting that the process of mESC tumor formation in vivo is potentially dependent on integrin interaction., Conclusions: Overall, the disruption of cell-integrin interaction via cRGDfC peptide can mimic the effect of mechanical stimulation on mESC pluripotency gene expression and also inhibit the tumorigenic potential of mESCs in vivo.

In embryonic development, cells differentiate through stereotypical sequences of intermediate states to generate particular mature fates. By contrast, driving differentiation by ectopically expressing terminal transcription factors (direct programming) can generate similar fates by alternative routes. How differentiation in direct programming relates to embryonic differentiation is unclear. We applied single-cell RNA sequencing to compare two motor neuron differentiation protocols: a standard protocol approximating the embryonic lineage, and a direct programming method. Both initially undergo similar early neural commitment. Later, the direct programming path diverges into a novel transitional state rather than following the expected embryonic spinal intermediates. The novel state in direct programming has specific and uncharacteristic gene expression. It forms a loop in gene expression space that converges separately onto the same final motor neuron state as the standard path. Despite their different developmental histories, motor neurons from both protocols structurally, functionally, and transcriptionally resemble motor neurons isolated from embryos.

Pluripotent stem cells can self-renew in culture and differentiate along all somatic lineages in vivo. While much is known about the molecular basis of pluripotency, the mechanisms of differentiation remain unclear. Here, we profile individual mouse embryonic stem cells as they progress along the neuronal lineage. We observe that cells pass from the pluripotent state to the neuronal state via an intermediate epiblast-like state. However, analysis of the rate at which cells enter and exit these observed cell states using a hidden Markov model indicates the presence of a chain of unobserved molecular states that each cell transits through stochastically in sequence. This chain of hidden states allows individual cells to record their position on the differentiation trajectory, thereby encoding a simple form of cellular memory. We suggest a statistical mechanics interpretation of these results that distinguishes between functionally distinct cellular "macrostates" and functionally similar molecular "microstates" and propose a model of stem cell differentiation as a non-Markov stochastic process., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Leveraging microfluidics and nano-plasmonics, we present in this paper a new method employing a micro-nano-device that is capable of monitoring the dynamic cell-substrate attachment process at single cell level in real time without labeling. The micro-nano-device essentially has a gold thin film as the substrate perforated with periodic, near-cm 2 -area, template-stripped nano-holes, which generate plasmonic extraordinary optical transmission (EOT) with a high sensitivity to refractive index changes at the metal-dielectric interface. Using this device, we successfully demonstrated label-free and real-time monitoring of the dynamic cell attachment process for single mouse embryonic stem cell (C3H10) and human tumor cell (HeLa) by collecting EOT spectrum data during 3-hour on-chip culture. We further collected the EOT spectral shift data at the start and end points of measurement during 3-hour on-chip culture for 50 C3H10 and 50 HeLa cells, respectively. The experiment results show that the single cell attachment process of both HeLa and C3H10 cells follow the logistic retarded growth model, but with different kinetic parameters. Variations in spectral shift during the same culture period across single cells present new evidence for cell heterogeneity. The micro-nano-device provides a new, label-free, real-time, and sensitive, platform to investigate the cell adhesion kinetics at single cell level.

Chromatin reorganization is necessary for pluripotent stem cells, including embryonic stem cells (ESCs), to acquire lineage potential. However, it remains unclear how ESCs maintain their characteristic chromatin state for appropriate gene expression upon differentiation. Here, we demonstrate that chromodomain helicase DNA-binding domain 2 (Chd2) is required to maintain the differentiation potential of mouse ESCs. Chd2-depleted ESCs showed suppressed expression of developmentally regulated genes upon differentiation and subsequent differentiation defects without affecting gene expression in the undifferentiated state. Furthermore, chromatin immunoprecipitation followed by sequencing revealed alterations in the nucleosome occupancy of the histone variant H3.3 for developmentally regulated genes in Chd2-depleted ESCs, which in turn led to elevated trimethylation of the histone H3 lysine 27. These results suggest that Chd2 is essential in preventing suppressive chromatin formation for developmentally regulated genes and determines subsequent effects on developmental processes in the undifferentiated state., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The capacity of pluripotent embryonic stem cells to differentiate into any cell type in the body makes them invaluable in the field of regenerative medicine. However, because of the complexity of both the core pluripotency network and the process of cell fate computation it is not yet possible to control the fate of stem cells. We present a theoretical model of stem cell fate computation that is based on Halley and Winkler's Branching Process Theory (BPT) and on Greaves et al.'s agent-based computer simulation derived from that theoretical model. BPT abstracts the complex production and action of a Transcription Factor (TF) into a single critical branching process that may dissipate, maintain, or become supercritical. Here we take the single TF model and extend it to multiple interacting TFs, and build an agent-based simulation of multiple TFs to investigate the dynamics of such coupled systems. We have developed the simulation and the theoretical model together, in an iterative manner, with the aim of obtaining a deeper understanding of stem cell fate computation, in order to influence experimental efforts, which may in turn influence the outcome of cellular differentiation. The model used is an example of self-organization and could be more widely applicable to the modelling of other complex systems. The simulation based on this model, though currently limited in scope in terms of the biology it represents, supports the utility of the Halley and Winkler branching process model in describing the behaviour of stem cell gene regulatory networks. Our simulation demonstrates three key features: (i) the existence of a critical value of the branching process parameter, dependent on the details of the cistrome in question; (ii) the ability of an active cistrome to "ignite" an otherwise fully dissipated cistrome, and drive it to criticality; (iii) how coupling cistromes together can reduce their critical branching parameter values needed to drive them to criticality.

The cell cycle has gained attention as a key determinant for cell fate decisions, but the contribution of DNA replication and mitosis in stem cell differentiation has not been extensively studied. To understand if these processes act as "windows of opportunity" for changes in cell identity, we established synchronized cultures of mouse embryonic stem cells as they exit the ground state of pluripotency. We show that initial transcriptional changes in this transition do not require passage through mitosis and that conversion to primed pluripotency is linked to lineage priming in the G1 phase. Importantly, we demonstrate that impairment of DNA replication severely blocks transcriptional switch to primed pluripotency, even in the absence of p53 activity induced by the DNA damage response. Our data suggest an important role for DNA replication during mouse embryonic stem cell differentiation, which could shed light on why pluripotent cells are only receptive to differentiation signals during G1, that is, before the S phase., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The recent development of haploid cell lines has facilitated forward genetic screenings in mammalian cells. These lines include near-haploid human cell lines isolated from a patient with chronic myelogenous leukemia (KBM7 and HAP1), as well as haploid embryonic stem cells derived from several organisms. In all cases, haploidy was shown to be an unstable state, so that cultures of mammalian haploid cells rapidly become enriched in diploids. Here we show that the observed diploidization is due to a proliferative disadvantage of haploid cells compared with diploid cells. Accordingly, single-cell-sorted haploid mammalian cells maintain the haploid state for prolonged periods, owing to the absence of competing diploids. Although the duration of interphase is similar in haploid and diploid cells, haploid cells spend longer in mitosis, indicative of problems in chromosome segregation. In agreement with this, a substantial proportion of the haploids die at or shortly after the last mitosis through activation of a p53-dependent cytotoxic response. Finally, we show that p53 deletion stabilizes haploidy in human HAP1 cells and haploid mouse embryonic stem cells. We propose that, similar to aneuploidy or tetraploidy, haploidy triggers a p53-dependent response that limits the fitness of mammalian cells., Competing Interests: The authors declare no conflict of interest.

Proper telomere length is essential for embryonic stem cell (ESC) self-renewal and pluripotency. Mouse ESCs (mESCs) sporadically convert to a transient totipotent state similar to that of two-cell (2C) embryos to recover shortened telomeres. Zscan4, which exhibits a burst of expression in 2C-like mESCs, is required for telomere extension in these cells. However, the mechanism by which Zscan4 extends telomeres remains elusive. Here, we show that Zscan4 facilitates telomere elongation by inducing global DNA demethylation through downregulation of Uhrf1 and Dnmt1, major components of the maintenance DNA methylation machinery. Mechanistically, Zscan4 recruits Uhrf1 and Dnmt1 and promotes their degradation, which depends on the E3 ubiquitin ligase activity of Uhrf1. Blocking DNA demethylation prevents telomere elongation associated with Zscan4 expression, suggesting that DNA demethylation mediates the effect of Zscan4. Our results define a molecular pathway that contributes to the maintenance of telomere length homeostasis in mESCs., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Execution of pluripotency requires progression from the naïve status represented by mouse embryonic stem cells (ESCs) to a state capacitated for lineage specification. This transition is coordinated at multiple levels. Non-coding RNAs may contribute to this regulatory orchestra. We identified a rodent-specific long non-coding RNA (lncRNA) linc1281, hereafter Ephemeron ( Eprn ), that modulates the dynamics of exit from naïve pluripotency. Eprn deletion delays the extinction of ESC identity, an effect associated with perduring Nanog expression. In the absence of Eprn , Lin28a expression is reduced which results in persistence of let-7 microRNAs, and the up-regulation of de novo methyltransferases Dnmt3a/b is delayed. Dnmt3a/b deletion retards ES cell transition, correlating with delayed Nanog promoter methylation and phenocopying loss of Eprn or Lin28a . The connection from lncRNA to miRNA and DNA methylation facilitates the acute extinction of naïve pluripotency, a pre-requisite for rapid progression from preimplantation epiblast to gastrulation in rodents. Eprn illustrates how lncRNAs may introduce species-specific network modulations.

In order to maintain tissue homeostasis and functionality, adherent cells need to sense and respond to environmental mechanical stimuli. An important ability that adherent cells need in order to properly sense and respond to mechanical stimuli is the ability to exert contractile stress onto the environment via actin stress fibers. The actin stress fibers form a structural chain between the cells' environment via focal adhesions and the nucleus via the nuclear lamina. In case one of the links in this chain is missing or aberrant, contractile stress generation will be affected. This is especially the case in laminopathic cells, which have a missing or mutated form of the LMNA gene encoding for part of the nuclear lamina. Using the thin film method combined with sample specific finite element modeling, we quantitatively showed a fivefold lower contractile stress generation of Lmna knockout mouse embryonic fibroblasts (MEFs) as compared to wild-type MEFs. Via fluorescence microscopy it was demonstrated that the lower contractile stress generation was associated with an impaired actin stress fiber organization with thinner actin fibers and smaller focal adhesions. Similar experiments with wild-type MEFs with chemically disrupted actin stress fibers verified these findings. These data illustrate the importance of an organized actin stress fiber network for contractile stress generation and demonstrate the devastating effect of an impaired stress fiber organization in laminopathic fibroblasts. Next to this, the thin film method is expected to be a promising tool in unraveling contractility differences between fibroblasts with different types of laminopathic mutations.

Mobilization of retrotransposons to new genomic locations is a significant driver of mammalian genome evolution, but these mutagenic events can also cause genetic disorders. In humans, retrotransposon mobilization is mediated primarily by proteins encoded by LINE-1 (L1) retrotransposons, which mobilize in pluripotent cells early in development. Here we show that TEX19.1, which is induced by developmentally programmed DNA hypomethylation, can directly interact with the L1-encoded protein L1-ORF1p, stimulate its polyubiquitylation and degradation, and restrict L1 mobilization. We also show that TEX19.1 likely acts, at least in part, through promoting the activity of the E3 ubiquitin ligase UBR2 towards L1-ORF1p. Moreover, loss of Tex19.1 increases L1-ORF1p levels and L1 mobilization in pluripotent mouse embryonic stem cells, implying that Tex19.1 prevents de novo retrotransposition in the pluripotent phase of the germline cycle. These data show that post-translational regulation of L1 retrotransposons plays a key role in maintaining trans-generational genome stability in mammals.

Embryonic stem (ES) cells go though embryo-like cell cycles regulated by specialized molecular mechanisms. However, it is not known whether there are ES cell-specific mechanisms regulating mitotic fidelity. Here we showed that Autoimmune Regulator ( Aire ), a transcription coordinator involved in immune tolerance processes, is a critical spindle-associated protein in mouse ES(mES) cells. BioID analysis showed that AIRE associates with spindle-associated proteins in mES cells. Loss of function analysis revealed that Aire was important for centrosome number regulation and spindle pole integrity specifically in mES cells. We also identified the c-terminal LESLL motif as a critical motif for AIRE's mitotic function. Combined maternal and zygotic knockout further revealed Aire's critical functions for spindle assembly in preimplantation embryos. These results uncovered a previously unappreciated function for Aire and provide new insights into the biology of stem cell proliferation and potential new angles to understand fertility defects in humans carrying Air e mutations.