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Development of embryonic stem cells (ESCs) into neurons requires intricate regulation of transcription, splicing, and translation, but how these processes interconnect is not understood. We found that polypyrimidine tract binding protein 1 (PTBP1) controls splicing of DPF2, a subunit of BRG1/BRM-associated factor (BAF) chromatin remodeling complexes. Dpf2 exon 7 splicing is inhibited by PTBP1 to produce the DPF2-S isoform early in development. During neuronal differentiation, loss of PTBP1 allows exon 7 inclusion and DPF2-L expression. Different cellular phenotypes and gene expression programs were induced by these alternative DPF2 isoforms. We identified chromatin binding sites enriched for each DPF2 isoform, as well as sites bound by both. In ESC, DPF2-S preferential sites were bound by pluripotency factors. In neuronal progenitors, DPF2-S sites were bound by nuclear factor I (NFI), while DPF2-L sites were bound by CCCTC-binding factor (CTCF). DPF2-S sites exhibited enhancer modifications, while DPF2-L sites showed promoter modifications. Thus, alternative splicing redirects BAF complex targeting to impact chromatin organization during neuronal development., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Post-transcriptional mechanisms have the potential to influence complex changes in gene expression, yet their role in cell fate transitions remains largely unexplored. Here, we show that suppression of the RNA helicase DDX6 endows human and mouse primed embryonic stem cells (ESCs) with a differentiation-resistant, "hyper-pluripotent" state, which readily reprograms to a naive state resembling the preimplantation embryo. We further demonstrate that DDX6 plays a key role in adult progenitors where it controls the balance between self-renewal and differentiation in a context-dependent manner. Mechanistically, DDX6 mediates the translational suppression of target mRNAs in P-bodies. Upon loss of DDX6 activity, P-bodies dissolve and release mRNAs encoding fate-instructive transcription and chromatin factors that re-enter the ribosome pool. Increased translation of these targets impacts cell fate by rewiring the enhancer, heterochromatin, and DNA methylation landscapes of undifferentiated cell types. Collectively, our data establish a link between P-body homeostasis, chromatin organization, and stem cell potency., (Copyright © 2019 Elsevier Inc. All rights reserved.)

FOXO3 is an evolutionarily conserved transcription factor that has been linked to longevity. Here we wanted to find out whether human vascular cells could be functionally enhanced by engineering them to express an activated form of FOXO3. This was accomplished via genome editing at two nucleotides in human embryonic stem cells, followed by differentiation into a range of vascular cell types. FOXO3-activated vascular cells exhibited delayed aging and increased resistance to oxidative injury compared with wild-type cells. When tested in a therapeutic context, FOXO3-enhanced vascular cells promoted vascular regeneration in a mouse model of ischemic injury and were resistant to tumorigenic transformation both in vitro and in vivo. Mechanistically, constitutively active FOXO3 conferred cytoprotection by transcriptionally downregulating CSRP1. Taken together, our findings provide mechanistic insights into FOXO3-mediated vascular protection and indicate that FOXO3 activation may provide a means for generating more effective and safe biomaterials for cell replacement therapies., (Copyright © 2018 Elsevier Inc. All rights reserved.)

BAF complexes are composed of different subunits with varying functional and developmental roles, although many subunits have not been examined in depth. Here we show that the Baf45 subunit Dpf2 maintains pluripotency and ESC differentiation potential. Dpf2 co-occupies enhancers with Oct4, Sox2, p300, and the BAF subunit Brg1, and deleting Dpf2 perturbs ESC self-renewal, induces repression of Tbx3, and impairs mesendodermal differentiation without dramatically altering Brg1 localization. Mesendodermal differentiation can be rescued by restoring Tbx3 expression, whose distal enhancer is positively regulated by Dpf2-dependent H3K27ac maintenance and recruitment of pluripotency TFs and Brg1. In contrast, the PRC2 subunit Eed binds an intragenic Tbx3 enhancer to oppose Dpf2-dependent Tbx3 expression and mesendodermal differentiation. The PRC2 subunit Ezh2 likewise opposes Dpf2-dependent differentiation through a distinct mechanism involving Nanog repression. Together, these findings delineate distinct mechanistic roles for specific BAF and PRC2 subunits during ESC differentiation., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

We report the direct reprogramming of both adult human fibroblasts and blood cells into induced neural plate border stem cells (iNBSCs) by ectopic expression of four neural transcription factors. Self-renewing, clonal iNBSCs can be robustly expanded in defined media while retaining multilineage differentiation potential. They generate functional cell types of neural crest and CNS lineages and could be used to model a human pain syndrome via gene editing of SCN9A in iNBSCs. NBSCs can also be derived from human pluripotent stem cells and share functional and molecular features with NBSCs isolated from embryonic day 8.5 (E8.5) mouse neural folds. Single-cell RNA sequencing identified the anterior hindbrain as the origin of mouse NBSCs, with human iNBSCs sharing a similar regional identity. In summary, we identify embryonic NBSCs and report their generation by direct reprogramming in human, which may facilitate insights into neural development and provide a neural stem cell source for applications in regenerative medicine., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Transcription factor (TF)-mediated reprogramming to pluripotency is a slow and inefficient process, because most pluripotency TFs fail to access relevant target sites in a refractory chromatin environment. It is still unclear how TFs actually orchestrate the opening of repressive chromatin during the long latency period of reprogramming. Here, we show that the orphan nuclear receptor Esrrb plays a pioneering role in recruiting the core pluripotency factors Oct4, Sox2, and Nanog to inactive enhancers in closed chromatin during the reprogramming of epiblast stem cells. Esrrb binds to silenced enhancers containing stable nucleosomes and hypermethylated DNA, which are inaccessible to the core factors. Esrrb binding is accompanied by local loss of DNA methylation, LIF-dependent engagement of p300, and nucleosome displacement, leading to the recruitment of core factors within approximately 2 days. These results suggest that TFs can drive rapid remodeling of the local chromatin structure, highlighting the remarkable plasticity of stable epigenetic information., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The embryonic stem cell (ESC) transition from naive to primed pluripotency is marked by major changes in cellular properties and developmental potential. ISY1 regulates microRNA (miRNA) biogenesis, yet its role and relevance to ESC biology remain unknown. Here, we find that highly dynamic ISY1 expression during the naive-to-primed ESC transition defines a specific phase of "poised" pluripotency characterized by distinct miRNA and mRNA transcriptomes and widespread poised cell contribution to mouse chimeras. Loss- and gain-of-function experiments reveal that ISY1 promotes exit from the naive state and is necessary and sufficient to induce and maintain poised pluripotency, and that persistent ISY1 overexpression inhibits the transition from the naive to the primed state. We identify a large subset of ISY1-dependent miRNAs that can rescue the inability of miRNA-deficient ESCs to establish the poised state and transition to the primed state. Thus, dynamic ISY1 regulates poised pluripotency through miRNAs to control ESC fate., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Stem cell-specific transcriptional networks are well known to control pluripotency, but constitutive cellular processes such as mRNA splicing and protein synthesis can add complex layers of regulation with poorly understood effects on cell-fate decisions. Here, we show that the RNA binding protein HTATSF1 controls embryonic stem cell differentiation by regulating multiple aspects of RNA processing during ribosome biogenesis. HTATSF1, in a complex with splicing factor SF3B1, controls intron removal from ribosomal protein transcripts and regulates ribosomal RNA transcription and processing, thereby controlling 60S ribosomal abundance and protein synthesis. HTATSF1-dependent protein synthesis is essential for naive pre-implantation epiblast to transition into post-implantation epiblast, a stage with transiently low protein synthesis, and further differentiation toward neuroectoderm. Together, these results identify coordinated regulation of ribosomal RNA and protein synthesis by HTATSF1 and show that this essential mechanism controls protein synthesis during early mammalian embryogenesis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

A permissive chromatin environment coupled to hypertranscription drives the rapid proliferation of embryonic stem cells (ESCs) and peri-implantation embryos. We carried out a genome-wide screen to systematically dissect the regulation of the euchromatic state of ESCs. The results revealed that cellular growth pathways, most prominently translation, perpetuate the euchromatic state and hypertranscription of ESCs. Acute inhibition of translation rapidly depletes euchromatic marks in mouse ESCs and blastocysts, concurrent with delocalization of RNA polymerase II and reduction in nascent transcription. Translation inhibition promotes rewiring of chromatin accessibility, which decreases at a subset of active developmental enhancers and increases at histone genes and transposable elements. Proteome-scale analyses revealed that several euchromatin regulators are unstable proteins and continuously depend on a high translational output. We propose that this mechanistic interdependence of euchromatin, transcription, and translation sets the pace of proliferation at peri-implantation and may be employed by other stem/progenitor cells., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Erasure of DNA methylation and repressive chromatin marks in the mammalian germline leads to risk of transcriptional activation of transposable elements (TEs). Here, we used mouse embryonic stem cells (ESCs) to identify an endosiRNA-based mechanism involved in suppression of TE transcription. In ESCs with DNA demethylation induced by acute deletion of Dnmt1, we saw an increase in sense transcription at TEs, resulting in an abundance of sense/antisense transcripts leading to high levels of ARGONAUTE2 (AGO2)-bound small RNAs. Inhibition of Dicer or Ago2 expression revealed that small RNAs are involved in an immediate response to demethylation-induced transposon activation, while the deposition of repressive histone marks follows as a chronic response. In vivo, we also found TE-specific endosiRNAs present during primordial germ cell development. Our results suggest that antisense TE transcription is a "trap" that elicits an endosiRNA response to restrain acute transposon activity during epigenetic reprogramming in the mammalian germline., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Blastocyst-derived embryonic stem cells (ESCs) and gonad-derived embryonic germ cells (EGCs) represent two classic types of pluripotent cell lines, yet their molecular equivalence remains incompletely understood. Here, we compare genome-wide methylation patterns between isogenic ESC and EGC lines to define epigenetic similarities and differences. Surprisingly, we find that sex rather than cell type drives methylation patterns in ESCs and EGCs. Cell fusion experiments further reveal that the ratio of X chromosomes to autosomes dictates methylation levels, with female hybrids being hypomethylated and male hybrids being hypermethylated. We show that the X-linked MAPK phosphatase DUSP9 is upregulated in female compared to male ESCs, and its heterozygous loss in female ESCs leads to male-like methylation levels. However, male and female blastocysts are similarly hypomethylated, indicating that sex-specific methylation differences arise in culture. Collectively, our data demonstrate the epigenetic similarity of sex-matched ESCs and EGCs and identify DUSP9 as a regulator of female-specific hypomethylation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Active DNA demethylation in mammals involves ten-eleven translocation (TET) family protein-mediated oxidation of 5-methylcytosine (5mC). However, base-resolution landscapes of 5-formylcytosine (5fC) (an oxidized derivative of 5mC) at the single-cell level remain unexplored. Here, we present "CLEVER-seq" (chemical-labeling-enabled C-to-T conversion sequencing), which is a single-cell, single-base resolution 5fC-sequencing technology, based on biocompatible, selective chemical labeling of 5fC and subsequent C-to-T conversion during amplification and sequencing. CLEVER-seq shows intrinsic 5fC heterogeneity in mouse early embryos, Epi stem cells (EpiSCs), and embryonic stem cells (ESCs). CLEVER-seq of mouse early embryos also reveals the highly patterned genomic distribution and parental-specific dynamics of 5fC during mouse early pre-implantation development. Integrated analysis demonstrates that promoter 5fC production precedes the expression upregulation of a clear set of developmentally and metabolically critical genes. Collectively, our work reveals the dynamics of active DNA demethylation during mouse pre-implantation development and provides an important resource for further functional studies of epigenetic reprogramming in single cells., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Poised enhancers marked by H3K27me3 in pluripotent stem cells have been implicated in the establishment of somatic expression programs during embryonic stem cell (ESC) differentiation. However, the functional relevance and mechanism of action of poised enhancers remain unknown. Using CRISPR/Cas9 technology to engineer precise genetic deletions, we demonstrate that poised enhancers are necessary for the induction of major anterior neural regulators. Interestingly, circularized chromosome conformation capture sequencing (4C-seq) shows that poised enhancers already establish physical interactions with their target genes in ESCs in a polycomb repressive complex 2 (PRC2)-dependent manner. Loss of PRC2 does not activate poised enhancers or induce their putative target genes in undifferentiated ESCs; however, loss of PRC2 in differentiating ESCs severely and specifically compromises the induction of major anterior neural genes representing poised enhancer targets. Overall, our work illuminates an unexpected function for polycomb proteins in facilitating neural induction by endowing major anterior neural loci with a permissive regulatory topology., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Direct cell programming via overexpression of transcription factors (TFs) aims to control cell fate with the degree of precision needed for clinical applications. However, the regulatory steps involved in successful terminal cell fate programming remain obscure. We have investigated the underlying mechanisms by looking at gene expression, chromatin states, and TF binding during the uniquely efficient Ngn2, Isl1, and Lhx3 motor neuron programming pathway. Our analysis reveals a highly dynamic process in which Ngn2 and the Isl1/Lhx3 pair initially engage distinct regulatory regions. Subsequently, Isl1/Lhx3 binding shifts from one set of targets to another, controlling regulatory region activity and gene expression as cell differentiation progresses. Binding of Isl1/Lhx3 to later motor neuron enhancers depends on the Ebf and Onecut TFs, which are induced by Ngn2 during the programming process. Thus, motor neuron programming is the product of two initially independent transcriptional modules that converge with a feedforward transcriptional logic., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Stem cell engineering and grafting of mesencephalic dopamine (mesDA) neurons is a promising strategy for brain repair in Parkinson's disease (PD). Refinement of differentiation protocols to optimize this approach will require deeper understanding of mesDA neuron development. Here, we studied this process using transcriptome-wide single-cell RNA sequencing of mouse neural progenitors expressing the mesDA neuron determinant Lmx1a. This approach resolved the differentiation of mesDA and neighboring neuronal lineages and revealed a remarkably close relationship between developing mesDA and subthalamic nucleus (STN) neurons, while also highlighting a distinct transcription factor set that can distinguish between them. While previous hESC mesDA differentiation protocols have relied on markers that are shared between the two lineages, we found that application of these highlighted markers can help to refine current stem cell engineering protocols, increasing the proportion of appropriately patterned mesDA progenitors. Our results, therefore, have important implications for cell replacement therapy in PD., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Hematopoietic stem and progenitor cell (HSPC) specification is regulated by numerous defined factors acting locally within the hemogenic niche; however, it is unclear whether production can adapt to fluctuating systemic needs. Here we show that the CNS controls embryonic HSPC numbers via the hypothalamic-pituitary-adrenal/interrenal (HPA/I) stress response axis. Exposure to serotonin or the reuptake inhibitor fluoxetine increased runx1 expression and Flk1(+)/cMyb(+) HSPCs independent of peripheral innervation. Inhibition of neuronal, but not peripheral, tryptophan hydroxlyase (Tph) persistently reduced HSPC number. Consistent with central HPA/I axis induction and glucocorticoid receptor (GR) activation, GR agonists enhanced, whereas GR loss diminished, HSPC formation. Significantly, developmental hypoxia, as indicated by Hif1α function, induced the HPA/I axis and cortisol production. Furthermore, Hif1α-stimulated HSPC enhancement was attenuated by neuronal tph or GR loss. Our data establish that embryonic HSC production responds to physiologic stress via CNS-derived serotonin synthesis and central feedback regulation to control HSC numbers., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Divergent lncRNAs that are transcribed in the opposite direction to nearby protein-coding genes comprise a significant proportion (∼20%) of total lncRNAs in mammalian genomes. Through genome-wide analysis, we found that the distribution of this lncRNA class strongly correlates with essential developmental regulatory genes. In pluripotent cells, divergent lncRNAs regulate the transcription of nearby genes. As an example, the divergent lncRNA Evx1as promotes transcription of its neighbor gene, EVX1, and regulates mesendodermal differentiation. At a single-cell level, early broad expression of Evx1as is followed by a rapid, high-level transcription of EVX1, supporting the idea that Evx1as plays an upstream role to facilitate EVX1 transcription. Mechanistically, Evx1as RNA binds to regulatory sites on chromatin, promotes an active chromatin state, and interacts with Mediator. Based on our analyses, we propose that the biological function of thousands of uncharacterized lncRNAs of this class may be inferred from the role of their neighboring adjacent genes., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Epigenetic and epitranscriptomic networks have important functions in maintaining the pluripotency of embryonic stem cells (ESCs) and somatic cell reprogramming. However, the mechanisms integrating the actions of these distinct networks are only partially understood. Here we show that the chromatin-associated zinc finger protein 217 (ZFP217) coordinates epigenetic and epitranscriptomic regulation. ZFP217 interacts with several epigenetic regulators, activates the transcription of key pluripotency genes, and modulates N6-methyladenosine (m(6)A) deposition on their transcripts by sequestering the enzyme m(6)A methyltransferase-like 3 (METTL3). Consistently, Zfp217 depletion compromises ESC self-renewal and somatic cell reprogramming, globally increases m(6)A RNA levels, and enhances m(6)A modification of the Nanog, Sox2, Klf4, and c-Myc mRNAs, promoting their degradation. ZFP217 binds its own target gene mRNAs, which are also METTL3 associated, and is enriched at promoters of m(6)A-modified transcripts. Collectively, these findings shed light on how a transcription factor can tightly couple gene transcription to m(6)A RNA modification to ensure ESC identity., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Induced pluripotent stem cells (iPSCs) undergo extensive nuclear reprogramming and are generally indistinguishable from embryonic stem cells (ESCs) in their functional capacity and transcriptome and DNA methylation profiles. However, direct conversion of cells from one lineage to another often yields incompletely reprogrammed, functionally compromised cells, raising the question of whether pluripotency is required to achieve a high degree of nuclear reprogramming. Here, we show that transient expression of Gata3, Eomes, and Tfap2c in mouse fibroblasts induces stable, transgene-independent trophoblast stem-like cells (iTSCs). iTSCs possess transcriptional profiles highly similar to blastocyst-derived TSCs, with comparable methylation and H3K27ac patterns and genome-wide H2A.X deposition. iTSCs generate trophoectodermal lineages upon differentiation, form hemorrhagic lesions, and contribute to developing placentas in chimera assays, indicating a high degree of nuclear reprogramming, with no evidence of passage through a transient pluripotent state. Together, these data demonstrate that extensive nuclear reprogramming can be achieved independently of pluripotency., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Because of their similarity to humans, non-human primates are important models for studying human disease and developing therapeutic strategies. Establishment of chimeric animals using embryonic stem cells (ESCs) could help with these investigations, but has not so far been achieved. Here, we show that cynomolgus monkey ESCs (cESCs) grown in adjusted culture conditions are able to incorporate into host embryos and develop into chimeras with contribution in all three germ layers and in germ cell progenitors. Under the optimized culture conditions, which are based on an approach developed previously for naive human ESCs, the cESCs displayed altered growth properties, gene expression profiles, and self-renewal signaling pathways, suggestive of an altered naive-like cell state. Thus our findings show that it is feasible to generate chimeric monkeys using ESCs and open up new avenues for the use of non-human primate models to study both pluripotency and human disease., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Cell-based therapies to treat retinal degeneration are now being tested in clinical trials. However, it is not known whether the source of stem cells is important for the production of differentiated cells suitable for transplantation. To test this, we generated induced pluripotent stem cells (iPSCs) from murine rod photoreceptors (r-iPSCs) and scored their ability to make retinae by using a standardized quantitative protocol called STEM-RET. We discovered that r-iPSCs more efficiently produced differentiated retinae than did embryonic stem cells (ESCs) or fibroblast-derived iPSCs (f-iPSCs). Retinae derived from f-iPSCs had fewer amacrine cells and other inner nuclear layer cells. Integrated epigenetic analysis showed that DNA methylation contributes to the defects in f-iPSC retinogenesis and that rod-specific CTCF insulator protein-binding sites may promote r-iPSC retinogenesis. Together, our data suggest that the source of stem cells is important for producing retinal neurons in three-dimensional (3D) organ cultures., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The relationship between chromatin organization and transcriptional regulation is an area of intense investigation. We characterized the spatial relationships between alleles of the Oct4, Sox2, and Nanog genes in single cells during the earliest stages of mouse embryonic stem cell (ESC) differentiation and during embryonic development. We describe homologous pairing of the Oct4 alleles during ESC differentiation and embryogenesis, and we present evidence that pairing is correlated with the kinetics of ESC differentiation. Importantly, we identify critical DNA elements within the Oct4 promoter/enhancer region that mediate pairing of Oct4 alleles. Finally, we show that mutation of OCT4/SOX2 binding sites within this region abolishes inter-chromosomal interactions and affects accumulation of the repressive H3K9me2 modification at the Oct4 enhancer. Our findings demonstrate that chromatin organization and transcriptional programs are intimately connected in ESCs and that the dynamic positioning of the Oct4 alleles is associated with the transition from pluripotency to lineage specification., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Pairing of homologous alleles is a phenomenon generally associated with imprinted and mono-allelically expressed loci. In this issue, Hogan et al. (2015) examine the earliest steps between pluripotency and lineage commitment in ESCs and find a critical role for transient pairing of Oct4 alleles in exiting the pluripotent state., (Copyright © 2015 Elsevier Inc. All rights reserved.)

About half of the human genome consists of highly repetitive elements, most of which are considered dispensable for human life. Here, we report that repetitive elements originating from endogenous retroviruses (ERVs) are systematically transcribed during human early embryogenesis in a stage-specific manner. Our analysis highlights that the long terminal repeats (LTRs) of ERVs provide the template for stage-specific transcription initiation, thereby generating hundreds of co-expressed, ERV-derived RNAs. Conversion of human embryonic stem cells (hESCs) to an epiblast-like state activates blastocyst-specific ERV elements, indicating that their activity dynamically reacts to changes in regulatory networks. In addition to initiating stage-specific transcription, many ERV families contain preserved splice sites that join the ERV segment with non-ERV exons in their genomic vicinity. In summary, we find that ERV expression is a hallmark of cellular identity and cell potency that characterizes the cell populations in early human embryos., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The generation of pluripotent stem cells by somatic cell nuclear transfer (SCNT) has recently been achieved in human cells and sparked new interest in this technology. The authors reporting this methodical breakthrough speculated that SCNT would allow the creation of patient-matched embryonic stem cells, even in patients with hereditary mitochondrial diseases. However, herein we show that mismatched mitochondria in nuclear-transfer-derived embryonic stem cells (NT-ESCs) possess alloantigenicity and are subject to immune rejection. In a murine transplantation setup, we demonstrate that allogeneic mitochondria in NT-ESCs, which are nucleus-identical to the recipient, may trigger an adaptive alloimmune response that impairs the survival of NT-ESC grafts. The immune response is adaptive, directed against mitochondrial content, and amenable for tolerance induction. Mitochondrial alloantigenicity should therefore be considered when developing therapeutic SCNT-based strategies., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Reactivation of the pluripotency network during somatic cell reprogramming by exogenous transcription factors involves chromatin remodeling and the recruitment of RNA polymerase II (Pol II) to target loci. Here, we report that Pol II is engaged at pluripotency promoters in reprogramming but remains paused and inefficiently released. We also show that bromodomain-containing protein 4 (BRD4) stimulates productive transcriptional elongation of pluripotency genes by dissociating the pause release factor P-TEFb from an inactive complex containing HEXIM1. Consequently, BRD4 overexpression enhances reprogramming efficiency and HEXIM1 suppresses it, whereas Brd4 and Hexim1 knockdown do the opposite. We further demonstrate that the reprogramming factor KLF4 helps recruit P-TEFb to pluripotency promoters. Our work thus provides a mechanism for explaining the reactivation of pluripotency genes in reprogramming and unveils an unanticipated role for KLF4 in transcriptional pause release., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Pluripotency is the remarkable capacity of a single cell to engender all the specialized cell types of an adult organism. This property can be captured indefinitely through derivation of self-renewing embryonic stem cells (ESCs), which represent an invaluable platform to investigate cell fate decisions and disease. Recent advances have revealed that manipulation of distinct signaling cues can render ESCs in a uniform "ground state" of pluripotency, which more closely recapitulates the pluripotent naive epiblast. Here we discuss the extrinsic and intrinsic regulatory principles that underpin the nature of pluripotency and consider the emerging spectrum of pluripotent states., (Copyright © 2014 Elsevier Inc. All rights reserved.)

For future application of induced pluripotent stem cell (iPSC) technology, the ability to assess the overall quality of iPSC clones will be an important issue. Here we show that the histone variant H2A.X is a functional marker that can distinguish the developmental potentials of mouse iPSC lines. We found that H2A.X is specifically targeted to and negatively regulates extraembryonic lineage gene expression in embryonic stem cells (ESCs) and prevents trophectoderm lineage differentiation. ESC-specific H2A.X deposition patterns are faithfully recapitulated in iPSCs that support the development of "all-iPS" animals via tetraploid complementation, the most stringent test available of iPSC quality. In contrast, iPSCs that fail to support all-iPS embryonic development show aberrant H2A.X deposition, upregulation of extraembryonic lineage genes, and a predisposition to extraembryonic differentiation. Thus, our work has highlighted an epigenetic mechanism for maintaining cell lineage commitment in ESCs and iPSCs that can be used to distinguish the quality of iPSC lines., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Induced pluripotent stem cells (iPSCs) are commonly generated by transduction of Oct4, Sox2, Klf4, and Myc (OSKM) into cells. Although iPSCs are pluripotent, they frequently exhibit high variation in terms of quality, as measured in mice by chimera contribution and tetraploid complementation. Reliably high-quality iPSCs will be needed for future therapeutic applications. Here, we show that one major determinant of iPSC quality is the combination of reprogramming factors used. Based on tetraploid complementation, we found that ectopic expression of Sall4, Nanog, Esrrb, and Lin28 (SNEL) in mouse embryonic fibroblasts (MEFs) generated high-quality iPSCs more efficiently than other combinations of factors including OSKM. Although differentially methylated regions, transcript number of master regulators, establishment of specific superenhancers, and global aneuploidy were comparable between high- and low-quality lines, aberrant gene expression, trisomy of chromosome 8, and abnormal H2A.X deposition were distinguishing features that could potentially also be applicable to human., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The maintenance of mouse embryonic stem cells (mESCs) requires LIF and serum. However, a pluripotent "ground state," bearing resemblance to preimplantation mouse epiblasts, can be established through dual inhibition (2i) of both prodifferentiation Mek/Erk and Gsk3/Tcf3 pathways. While Gsk3 inhibition has been attributed to the transcriptional derepression of Esrrb, the molecular mechanism mediated by Mek inhibition remains unclear. In this study, we show that Krüppel-like factor 2 (Klf2) is phosphorylated by Erk2 and that phospho-Klf2 is proteosomally degraded. Mek inhibition hence prevents Klf2 protein phosphodegradation to sustain pluripotency. Indeed, while Klf2-null mESCs can survive under LIF/Serum, they are not viable under 2i, demonstrating that Klf2 is essential for ground state pluripotency. Importantly, we also show that ectopic Klf2 expression can replace Mek inhibition in mESCs, allowing the culture of Klf2-null mESCs under Gsk3 inhibition alone. Collectively, our study defines the Mek/Erk/Klf2 axis that cooperates with the Gsk3/Tcf3/Esrrb pathway in mediating ground state pluripotency., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Naive mouse embryonic stem cells (mESCs) and primed epiblast stem cells (mEpiSCs) represent successive snapshots of pluripotency during embryogenesis. Using transcriptomic and epigenomic mapping we show that a small fraction of transcripts are differentially expressed between mESCs and mEpiSCs and that these genes show expected changes in chromatin at their promoters and enhancers. Unexpectedly, the cis-regulatory circuitry of genes that are expressed at identical levels between these cell states also differs dramatically. In mESCs, these genes are associated with dominant proximal enhancers and dormant distal enhancers, which we term seed enhancers. In mEpiSCs, the naive-dominant enhancers are lost, and the seed enhancers take up primary transcriptional control. Seed enhancers have increased sequence conservation and show preferential usage in downstream somatic tissues, often expanding into super enhancers. We propose that seed enhancers ensure proper enhancer utilization and transcriptional fidelity as mammalian cells transition from naive pluripotency to a somatic regulatory program., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Fine-tuning of the pluripotency program is executed by a multitude of cellular processes. Two recent studies published in Cell Stem Cell (Wang et al., 2013; Tahmasebi et al., 2014) provide novel insights into the posttranscriptional and translational regulatory mechanisms controlling stem cell pluripotency and somatic cell reprogramming., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The master transcription factors play integral roles in the pluripotency transcription circuitry of embryonic stem cells (ESCs). How they selectively activate expression of the pluripotency network while simultaneously repressing genes involved in differentiation is not fully understood. Here, we define a requirement for the INO80 complex, a SWI/SNF family chromatin remodeler, in ESC self-renewal, somatic cell reprogramming, and blastocyst development. We show that Ino80, the chromatin remodeling ATPase, co-occupies pluripotency gene promoters with the master transcription factors, and its occupancy is dependent on OCT4 and WDR5. At the pluripotency genes, Ino80 maintains an open chromatin architecture and licenses recruitment of Mediator and RNA polymerase II for gene activation. Our data reveal an essential role for INO80 in the expression of the pluripotency network and illustrate the coordination among chromatin remodeler, transcription factor, and histone-modifying enzyme in the regulation of the pluripotent state., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Pluripotent stem cells have distinct metabolic requirements, and reprogramming cells to pluripotency requires a shift from oxidative to glycolytic metabolism. Here, we show that this shift occurs early during reprogramming of human cells and requires hypoxia-inducible factors (HIFs) in a stage-specific manner. HIF1α and HIF2α are both necessary to initiate this metabolic switch and for the acquisition of pluripotency, and the stabilization of either protein during early phases of reprogramming is sufficient to induce the switch to glycolytic metabolism. In contrast, stabilization of HIF2α during later stages represses reprogramming, partly because of the upregulation of TNF-related apoptosis-inducing ligand (TRAIL). TRAIL inhibits induced pluripotent stem cell (iPSC) generation by repressing apoptotic caspase 3 activity specifically in cells undergoing reprogramming but not human embryonic stem cells (hESCs), and inhibiting TRAIL activity enhances human iPSC generation. These results shed light on the mechanisms underlying the metabolic shifts associated with the acquisition of a pluripotent identity during reprogramming., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Translational control plays a pivotal role in the regulation of the pluripotency network in embryonic stem cells, but its effect on reprogramming somatic cells to pluripotency has not been explored. Here, we show that eukaryotic translation initiation factor 4E (eIF4E) binding proteins (4E-BPs), which are translational repressors, have a multifaceted effect on the reprogramming of mouse embryonic fibroblasts (MEFs) into induced pluripotent stem cells (iPSCs). Loss of 4E-BP expression attenuates the induction of iPSCs at least in part through increased translation of p21, a known inhibitor of somatic cell reprogramming. However, MEFs lacking both p53 and 4E-BPs show greatly enhanced reprogramming resulting from a combination of reduced p21 transcription and enhanced translation of endogenous mRNAs such as Sox2 and Myc and can be reprogrammed through the expression of only exogenous Oct4. Thus, 4E-BPs exert both positive and negative effects on reprogramming, highlighting the key role that translational control plays in regulating this process., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The creation of induced pluripotent stem cells (iPSCs) from somatic cells by ectopic expression of transcription factors has galvanized the fields of regenerative medicine and developmental biology. Here, we report a kinome-wide RNAi-based analysis to identify kinases that regulate somatic cell reprogramming to iPSCs. We prepared 3,686 small hairpin RNA (shRNA) lentiviruses targeting 734 kinase genes covering the entire mouse kinome and individually examined their effects on iPSC generation. We identified 59 kinases as barriers to iPSC generation and characterized seven of them further. We found that shRNA-mediated knockdown of the serine/threonine kinases TESK1 or LIMK2 promoted mesenchymal-to-epithelial transition, decreased COFILIN phosphorylation, and disrupted Actin filament structures during reprogramming of mouse embryonic fibroblasts. Similarly, knockdown of TESK1 in human fibroblasts also promoted reprogramming to iPSCs. Our study reveals the breadth of kinase networks regulating pluripotency and identifies a role for cytoskeletal remodeling in modulating the somatic cell reprogramming process., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The donor-dependent supply of platelets is frequently insufficient to meet transfusion needs. To address this issue, we developed a clinically applicable strategy for the derivation of functional platelets from human pluripotent stem cells (PSCs). This approach involves the establishment of stable immortalized megakaryocyte progenitor cell lines (imMKCLs) from PSC-derived hematopoietic progenitors through the overexpression of BMI1 and BCL-XL to respectively suppress senescence and apoptosis and the constrained overexpression of c-MYC to promote proliferation. The resulting imMKCLs can be expanded in culture over extended periods (4-5 months), even after cryopreservation. Halting the overexpression of c-MYC, BMI1, and BCL-XL in growing imMKCLs led to the production of CD42b(+) platelets with functionality comparable to that of native platelets on the basis of a range of assays in vitro and in vivo. The combination of robust expansion capacity and efficient platelet production means that appropriately selected imMKCL clones represent a potentially inexhaustible source of hPSC-derived platelets for clinical application., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Tet-mediated DNA oxidation is a recently identified mammalian epigenetic modification, and its functional role in cell-fate transitions remains poorly understood. Here, we derive mouse embryonic fibroblasts (MEFs) deleted in all three Tet genes and examine their capacity for reprogramming into induced pluripotent stem cells (iPSCs). We show that Tet-deficient MEFs cannot be reprogrammed because of a block in the mesenchymal-to-epithelial transition (MET) step. Reprogramming of MEFs deficient in TDG is similarly impaired. The block in reprogramming is caused at least in part by defective activation of key miRNAs, which depends on oxidative demethylation promoted by Tet and TDG. Reintroduction of either the affected miRNAs or catalytically active Tet and TDG restores reprogramming in the knockout MEFs. Thus, oxidative demethylation to promote gene activation appears to be functionally required for reprogramming of fibroblasts to pluripotency. These findings provide mechanistic insight into the role of epigenetic barriers in cell-lineage conversion., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Recent advances in embryonic stem cell (ESC) derivation and genome editing offer efficient platforms for genetic screening. In this issue, Li et al. and Leeb et al., respectively, expand such applications by generating haploid rat ESCs for screening, mutagenesis, and CRISPR-Cas-mediated gene targeting and by developing a forward genetic screen for interrogating haploid mESCs., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Self-renewal circuitry in embryonic stem cells (ESCs) is increasingly defined. How the robust pluripotency program is dissolved to enable fate transition is less appreciated. Here we develop a forward genetic approach using haploid ESCs. We created libraries of transposon integrations and screened for persistent self-renewal in differentiation-permissive culture. This yielded multiple mutants in the Fgf/Erk and GSK3/Tcf3 modules known to drive differentiation and in epigenetic modifiers implicated in lineage commitment. We also identified and validated factors not previously considered. These include the conserved small zinc finger protein Zfp706 and the RNA binding protein Pum1. Pum1 targets several mRNAs for naive pluripotency transcription factors and accelerates their downregulation at the onset of differentiation. These findings indicate that the dismantling of pluripotent circuitry proceeds at multiple levels. More broadly they exemplify the power of haploid ESCs for genetic interrogation of developmental processes., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The rat is an important animal model in biomedical research, but practical limitations to genetic manipulation have restricted the application of genetic analysis. Here we report the derivation of rat androgenetic haploid embryonic stem cells (RahESCs) as a tool to facilitate such studies. Our approach is based on removal of the maternal pronucleus from zygotes to generate androgenetic embryos followed by derivation of ESCs. The resulting RahESCs have 21 chromosomes, express pluripotency markers, differentiate into three germ layer cells, and contribute to the germline. Homozygous mutations can be introduced by both large-scale gene trapping and precise gene targeting via homologous recombination or the CRISPR-Cas system. RahESCs can also produce fertile rats after intracytoplasmic injection into oocytes and are therefore able to transmit genetic modifications to offspring. Overall, RahESCs represent a practical tool for functional genetic studies and production of transgenic lines in rat., (Copyright © 2014 Elsevier Inc. All rights reserved.)

During early development of female mouse embryos, both X chromosomes are transiently active. X gene dosage is then equalized between the sexes through the process of X chromosome inactivation (XCI). Whether the double dose of X-linked genes in females compared with males leads to sex-specific developmental differences has remained unclear. Using embryonic stem cells with distinct sex chromosome compositions as a model system, we show that two X chromosomes stabilize the naive pluripotent state by inhibiting MAPK and Gsk3 signaling and stimulating the Akt pathway. Since MAPK signaling is required to exit the pluripotent state, differentiation is paused in female cells as long as both X chromosomes are active. By preventing XCI or triggering it precociously, we demonstrate that this differentiation block is released once XX cells have undergone X inactivation. We propose that double X dosage interferes with differentiation, thus ensuring a tight coupling between X chromosome dosage compensation and development., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Human pluripotent stem cell (hPSC) differentiation typically yields heterogeneous populations. Knowledge of signals controlling embryonic lineage bifurcations could efficiently yield desired cell types through exclusion of alternate fates. Therefore, we revisited signals driving induction and anterior-posterior patterning of definitive endoderm to generate a coherent roadmap for endoderm differentiation. With striking temporal dynamics, BMP and Wnt initially specified anterior primitive streak (progenitor to endoderm), yet, 24 hr later, suppressed endoderm and induced mesoderm. At lineage bifurcations, cross-repressive signals separated mutually exclusive fates; TGF-β and BMP/MAPK respectively induced pancreas versus liver from endoderm by suppressing the alternate lineage. We systematically blockaded alternate fates throughout multiple consecutive bifurcations, thereby efficiently differentiating multiple hPSC lines exclusively into endoderm and its derivatives. Comprehensive transcriptional and chromatin mapping of highly pure endodermal populations revealed that endodermal enhancers existed in a surprising diversity of "pre-enhancer" states before activation, reflecting the establishment of a permissive chromatin landscape as a prelude to differentiation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Although somatic cell nuclear transfer (SCNT) and induction of pluripotency (to form iPSCs) are both recognized reprogramming methods, there has been relatively little comparative analysis of the resulting pluripotent cells. Here, we examine the capacity of these two reprogramming approaches to rejuvenate telomeres using late-generation telomerase-deficient (Terc(-/-)) mice that exhibit telomere dysfunction and premature aging. We found that embryonic stem cells established from Terc(-/-) SCNT embryos (Terc(-/-) ntESCs) have greater differentiation potential and self-renewal capacity than Terc(-/-) iPSCs. Remarkably, SCNT results in extensive telomere lengthening in cloned embryos and improved telomere capping function in the established Terc(-/-) ntESCs. In addition, mitochondrial function is severely impaired in Terc(-/-) iPSCs and their differentiated derivatives but significantly improved in Terc(-/-) ntESCs. Thus, our results suggest that SCNT-mediated reprogramming mitigates telomere dysfunction and mitochondrial defects to a greater extent than iPSC-based reprogramming. Understanding the basis of this differential could help optimize reprogramming strategies., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Human embryonic stem cells (hESCs) hold great promise for cell therapy as a source of diverse differentiated cell types. One key bottleneck to realizing such potential is allogenic immune rejection of hESC-derived cells by recipients. Here, we optimized humanized mice (Hu-mice) reconstituted with a functional human immune system that mounts a vigorous rejection of hESCs and their derivatives. We established knockin hESCs that constitutively express CTLA4-Ig and PD-L1 before and after differentiation, denoted CP hESCs. We then demonstrated that allogenic CP hESC-derived teratomas, fibroblasts, and cardiomyocytes are immune protected in Hu-mice, while cells derived from parental hESCs are effectively rejected. Expression of both CTLA4-Ig, which disrupts T cell costimulatory pathways, and PD-L1, which activates T cell inhibitory pathway, is required to confer immune protection, as neither was sufficient on their own. These findings are instrumental for developing a strategy to protect hESC-derived cells from allogenic immune responses without requiring systemic immune suppression., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Mouse epiblast stem cells (EpiSCs) can be derived from a wide range of developmental stages. To characterize and compare EpiSCs with different origins, we derived a series of EpiSC lines from pregastrula stage to late-bud-stage mouse embryos. We found that the transcriptomes of these cells are hierarchically distinct from those of the embryonic stem cells, induced pluripotent stem cells (iPSCs), and epiblast/ectoderm. The EpiSCs display globally similar gene expression profiles irrespective of the original developmental stage of the source tissue. They are developmentally similar to the ectoderm of the late-gastrula-stage embryo and behave like anterior primitive streak cells when differentiated in vitro and in vivo. The EpiSC lines that we derived can also be categorized based on a correlation between gene expression signature and predisposition to differentiate into particular germ-layer derivatives. Our findings therefore highlight distinct identifying characteristics of EpiSCs and provide a foundation for further examination of EpiSC properties and potential., (Copyright © 2014 Elsevier Inc. All rights reserved.)

How TGF-β signaling switches from enforcing pluripotency to promoting mesendodermal differentiation remains an open question. Recently in Cell Reports, Beyer et al. demonstrated that Hippo signaling components recruit the NuRD complex to repress expression of key genes targeted by TGF-β and thus determine whether TGF-β signaling will favor pluripotency or differentiation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Recapitulating three-dimensional (3D) structures of complex organs, such as the kidney, from pluripotent stem cells (PSCs) is a major challenge. Here, we define the developmental origins of the metanephric mesenchyme (MM), which generates most kidney components. Unexpectedly, we find that posteriorly located T(+) MM precursors are developmentally distinct from Osr1(+) ureteric bud progenitors during the postgastrulation stage, and we identify phasic Wnt stimulation and stage-specific growth factor addition as molecular cues that promote their development into the MM. We then use this information to derive MM from PSCs. These progenitors reconstitute the 3D structures of the kidney in vitro, including glomeruli with podocytes and renal tubules with proximal and distal regions and clear lumina. Furthermore, the glomeruli are efficiently vascularized upon transplantation. Thus, by reevaluating the developmental origins of metanephric progenitors, we have provided key insights into kidney specification in vivo and taken important steps toward kidney organogenesis in vitro., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Human embryonic stem cells (hESCs) are derived from the inner cell mass of the blastocyst. Despite sharing the common property of pluripotency, hESCs are notably distinct from epiblast cells of the preimplantation blastocyst. Here we use a combination of three small-molecule inhibitors to sustain hESCs in a LIF signaling-dependent hESC state (3iL hESCs) with elevated expression of NANOG and epiblast-enriched genes such as KLF4, DPPA3, and TBX3. Genome-wide transcriptome analysis confirms that the expression signature of 3iL hESCs shares similarities with native preimplantation epiblast cells. We also show that 3iL hESCs have a distinct epigenetic landscape, characterized by derepression of preimplantation epiblast genes. Using genome-wide binding profiles of NANOG and OCT4, we identify enhancers that contribute to rewiring of the regulatory circuitry. In summary, our study identifies a distinct hESC state with defined regulatory circuitry that will facilitate future analysis of human preimplantation embryogenesis and pluripotency., (Copyright © 2013 Elsevier Inc. All rights reserved.)