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Embryonic stem (ES) cells can undergo many aspects of mammalian embryogenesis in vitro1–5, but their developmental potential is substantially extended by interactions with extraembryonic stem cells, including trophoblast stem (TS) cells, extraembryonic endoderm stem (XEN) cells and inducible XEN (iXEN) cells6–11. Here we assembled stem cell-derived embryos in vitro from mouse ES cells, TS cells and iXEN cells and showed that they recapitulate the development of whole natural mouse embryo in utero up to day 8.5 post-fertilization. Our embryo model displays headfolds with defined forebrain and midbrain regions and develops a beating heart-like structure, a trunk comprising a neural tube and somites, a tail bud containing neuromesodermal progenitors, a gut tube, and primordial germ cells. This complete embryo model develops within an extraembryonic yolk sac that initiates blood island development. Notably, we demonstrate that the neurulating embryo model assembled from Pax6-knockout ES cells aggregated with wild-type TS cells and iXEN cells recapitulates the ventral domain expansion of the neural tube that occurs in natural, ubiquitous Pax6-knockout embryos. Thus, these complete embryoids are a powerful in vitro model for dissecting the roles of diverse cell lineages and genes in development. Our results demonstrate the self-organization ability of ES cells and two types of extraembryonic stem cells to reconstitute mammalian development through and beyond gastrulation to neurulation and early organogenesis.

Cell asked LGBTQ+ scientists around the world about how their identity shapes their experiences in STEM. Here we share six unique perspectives of researchers highlighting how their area of expertise, research focus, institutions, and geographical location have played a role in this regard. We thank them for sharing their voices and continued efforts toward making science more inclusive.

The scarcity of donor organs may be addressed in the future by using pigs to grow humanized organs with lower potential for immunological rejection after transplantation in humans. Previous studies have demonstrated that interspecies complementation of rodent blastocysts lacking a developmental regulatory gene can generate xenogeneic pancreas and kidney1,2. However, such organs contain host endothelium, a source of immune rejection. We used gene editing and somatic cell nuclear transfer to engineer porcine embryos deficient in ETV2, a master regulator of hematoendothelial lineages3-7. ETV2-null pig embryos lacked hematoendothelial lineages and were embryonic lethal. Blastocyst complementation with wild-type porcine blastomeres generated viable chimeric embryos whose hematoendothelial cells were entirely donor-derived. ETV2-null blastocysts were injected with human induced pluripotent stem cells (hiPSCs) or hiPSCs overexpressing the antiapoptotic factor BCL2, transferred to synchronized gilts and analyzed between embryonic day 17 and embryonic day 18. In these embryos, all endothelial cells were of human origin.

In vitro cultured stem cells with distinct developmental capacities can contribute to embryonic or extraembryonic tissues after microinjection into pre-implantation mammalian embryos. However, whether cultured stem cells can independently give rise to entire gastrulating embryo-like structures with embryonic and extraembryonic compartments remains unknown. Here, we adapt a recently established platform for prolonged ex utero growth of natural embryos to generate mouse post-gastrulation synthetic whole embryo models (sEmbryos), with both embryonic and extraembryonic compartments, starting solely from naive ESCs. This was achieved by co-aggregating non-transduced ESCs, with naive ESCs transiently expressing Cdx2 or Gata4 to promote their priming toward trophectoderm and primitive endoderm lineages, respectively. sEmbryos adequately accomplish gastrulation, advance through key developmental milestones, and develop organ progenitors within complex extraembryonic compartments similar to E8.5 stage mouse embryos. Our findings highlight the plastic potential of naive pluripotent cells to self-organize and functionally reconstitute and model the entire mammalian embryo beyond gastrulation.

Antibody-mediated immunity is initiated by B cell differentiation into multiple cell subsets, including plasmablast, memory, and germinal center (GC) cells. B cell differentiation trajectories are determined by transcription factors, yet very few mechanisms that specifically determine early B cell fates have been described. Here, we report a post-transcriptional mechanism that suppresses the plasmablast genetic program and promotes GC B cell fate commitment. Single-cell RNA-sequencing analysis reveals that antigen-specific B cell precursors at the pre-GC stage upregulate YTHDF2, which enhances the decay of methylated transcripts. Ythdf2-deficient B cells exhibit intact proliferation and activation, whereas differentiation into GC B cells is blocked. Mechanistically, B cells require YTHDF2 to attenuate the plasmablast genetic program during GC seeding, and transcripts of key plasmablast-regulating genes are methylated and bound by YTHDF2. Collectively, this study reveals how post-transcriptional suppression of gene expression directs appropriate B cell fate commitment during initiation of the adaptive immune response.

Research on early postimplantation mammalian development has been limited by the small size and intrauterine confinement of the developing embryos. Owing to the inability to observe and manipulate living embryos at these stages in utero, the establishment of robust ex utero embryo-culture systems that capture prolonged periods of mouse development has been an important research goal. In the last few years, these methods have been significantly improved by the optimization and enhancement of in vitro culture systems sustaining embryo development during peri-implantation stages for several species, and more recently, proper growth of natural mouse embryos from pregastrulation to late organogenesis stages and of embryonic stem cell (ES)-derived synthetic embryo models until early organogenesis stages. Here, we discuss the most recent ex utero embryo-culture systems established to date for rodents, nonhuman primates, and humans. We emphasize their technical aspects and developmental timeframe and provide insights into the new opportunities that these methods will contribute to the study of natural and synthetic mammalian embryogenesis and the stem-cell field.

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. Platforms have been recently developed for highly robust and prolonged ex utero culture of mouse embryos from egg-cylinder stages until advanced organogenesis. These platforms enable appropriate and faithful development of pregastrulating embryos (E5.5) until the hind limb formation stage (E11). Late gastrulating embryos (E7.5) are grown in rotating bottles in these settings, while extended culture from pregastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle cultures. In addition, sensitive regulation of O2 and CO2 concentration, gas pressure, glucose levels, and the use of a specific ex utero culture medium are critical for proper embryo development. Here, a detailed step-by-step protocol for extended ex utero mouse embryo culture is provided. The ability to grow normal mouse embryos ex utero from gastrulation to organogenesis represents a valuable tool for characterizing the effect of different experimental perturbations during embryonic development.

Developmental and epileptic encephalopathies (DEE) are a group of disorders associated with intractable seizures, brain development, and functional abnormalities, and in some cases, premature death. Pathogenic human germline biallelic mutations in tumor suppressor WW domain‐containing oxidoreductase (WWOX) are associated with a relatively mild autosomal recessive spinocerebellar ataxia‐12 (SCAR12) and a more severe early infantile WWOX‐related epileptic encephalopathy (WOREE). In this study, we generated an in vitro model for DEEs, using the devastating WOREE syndrome as a prototype, by establishing brain organoids from CRISPR‐engineered human ES cells and from patient‐derived iPSCs. Using these models, we discovered dramatic cellular and molecular CNS abnormalities, including neural population changes, cortical differentiation malfunctions, and Wnt pathway and DNA damage response impairment. Furthermore, we provide a proof of concept that ectopic WWOX expression could potentially rescue these phenotypes. Our findings underscore the utility of modeling childhood epileptic encephalopathies using brain organoids and their use as a unique platform to test possible therapeutic intervention strategies., Mutations in the human WWOX gene cause devastating developmental and neurological diseases in young children called WOREE syndrome and SCAR12 syndrome. Using both gene editing and reprogramming technologies these maladies can now be modeled in human brain organoids, allowing for molecular and electrophysiological study of the pathology, together with testing possible therapeutic interventions.

Supplemental Digital Content is available in the text., Background: N6-Methyladenosine (m6A) methylation is the most prevalent internal posttranscriptional modification on mammalian mRNA. The role of m6A mRNA methylation in the heart is not known. Methods: To determine the role of m6A methylation in the heart, we isolated primary cardiomyocytes and performed m6A immunoprecipitation followed by RNA sequencing. We then generated genetic tools to modulate m6A levels in cardiomyocytes by manipulating the levels of the m6A RNA methylase methyltransferase-like 3 (METTL3) both in culture and in vivo. We generated cardiac-restricted gain- and loss-of-function mouse models to allow assessment of the METTL3-m6A pathway in cardiac homeostasis and function. Results: We measured the level of m6A methylation on cardiomyocyte mRNA, and found a significant increase in response to hypertrophic stimulation, suggesting a potential role for m6A methylation in the development of cardiomyocyte hypertrophy. Analysis of m6A methylation showed significant enrichment in genes that regulate kinases and intracellular signaling pathways. Inhibition of METTL3 completely abrogated the ability of cardiomyocytes to undergo hypertrophy when stimulated to grow, whereas increased expression of the m6A RNA methylase METTL3 was sufficient to promote cardiomyocyte hypertrophy both in vitro and in vivo. Finally, cardiac-specific METTL3 knockout mice exhibit morphological and functional signs of heart failure with aging and stress, showing the necessity of RNA methylation for the maintenance of cardiac homeostasis. Conclusions: Our study identified METTL3-mediated methylation of mRNA on N6-adenosines as a dynamic modification that is enhanced in response to hypertrophic stimuli and is necessary for a normal hypertrophic response in cardiomyocytes. Enhanced m6A RNA methylation results in compensated cardiac hypertrophy, whereas diminished m6A drives eccentric cardiomyocyte remodeling and dysfunction, highlighting the critical importance of this novel stress-response mechanism in the heart for maintaining normal cardiac function.

N6-methyladenosine (m6A) is the most common mRNA modification. Recent studies have revealed that depletion of m6A machinery leads to alterations in the propagation of diverse viruses. These effects were proposed to be mediated through dysregulated methylation of viral RNA. Here we show that following viral infection or stimulation of cells with an inactivated virus, deletion of the m6A 'writer' METTL3 or 'reader' YTHDF2 led to an increase in the induction of interferon-stimulated genes. Consequently, propagation of different viruses was suppressed in an interferon-signaling-dependent manner. Significantly, the mRNA of IFNB, the gene encoding the main cytokine that drives the type I interferon response, was m6A modified and was stabilized following repression of METTL3 or YTHDF2. Furthermore, we show that m6A-mediated regulation of interferon genes was conserved in mice. Together, our findings uncover the role m6A serves as a negative regulator of interferon response by dictating the fast turnover of interferon mRNAs and consequently facilitating viral propagation.

The role of mitochondria dynamics and its molecular regulators remains largely unknown during naïve-to-primed pluripotent cell interconversion. Here we report that mitochondrial MTCH2 is a regulator of mitochondrial fusion, essential for the naïve-to-primed interconversion of murine embryonic stem cells (ESCs). During this interconversion, wild-type ESCs elongate their mitochondria and slightly alter their glutamine utilization. In contrast, MTCH2−/− ESCs fail to elongate their mitochondria and to alter their metabolism, maintaining high levels of histone acetylation and expression of naïve pluripotency markers. Importantly, enforced mitochondria elongation by the pro-fusion protein Mitofusin (MFN) 2 or by a dominant negative form of the pro-fission protein dynamin-related protein (DRP) 1 is sufficient to drive the exit from naïve pluripotency of both MTCH2−/− and wild-type ESCs. Taken together, our data indicate that mitochondria elongation, governed by MTCH2, plays a critical role and constitutes an early driving force in the naïve-to-primed pluripotency interconversion of murine ESCs., Reprogramming of mitochondria metabolism occurs during naïve to primed pluripotency differentiation in mouse embryonic stem cells (ESCs). Here the authors show that mitochondrial MTCH2 regulates mitochondrial fusion and that this fusion is required for naïve to primed pluripotency conversion

In mammals, morphogenesis and organogenesis take place after the embryo implants into the uterus, which makes it relatively inaccessible for observation and manipulation. While methods for in vitro culture of pre- and peri-implantation mouse embryos are routinely used, approaches for efficient and stable culture of post-implantation embryos from egg cylinder stages until advanced organogenesis remain to be established. We recently developed highly robust ex utero post-implantation mouse embryo culture platforms, that enable appropriate and faithful development of embryos before gastrulation (E5.5) until the hind limb formation stage (E11). In these protocols, late gastrulating embryos (E7.5) are grown in 3D rotating bottles settings, while extended culture from pre-gastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle culture protocols. These systems support stable growth of normal mouse embryos ex utero from pre-gastrulation to advanced organogenesis.

Long-lasting immunity depends on the generation of protective antibodies through the germinal center (GC) reaction. N6-methyladenosine (m6A) modification of mRNAs by METTL3 activity modulates transcript lifetime primarily through the function of m6A readers; however, the physiological role of this molecular machinery in the GC remains unknown. Here, we show that m6A modifications by METTL3 are required for GC maintenance through the differential functions of m6A readers. Mettl3-deficient GC B cells exhibited reduced cell-cycle progression and decreased expression of proliferation- and oxidative phosphorylation–related genes. The m6A binder, IGF2BP3, was required for stabilization of Myc mRNA and expression of its target genes, whereas the m6A reader, YTHDF2, indirectly regulated the expression of the oxidative phosphorylation gene program. Our findings demonstrate how two independent gene networks that support critical GC functions are modulated by m6A through distinct mRNA binders.

The fidelity of the early embryonic program is underlined by tight regulation of the chromatin. Yet, how the chromatin is organized to prohibit the reversal of the developmental program remains unclear. Specifically, the totipotency-to-pluripotency transition marks one of the most dramatic events to the chromatin, and yet, the nature of histone alterations underlying this process is incompletely characterized. Here, we show that linker histone H1 is post-translationally modulated by SUMO2/3, which facilitates its fixation onto ultra-condensed heterochromatin in embryonic stem cells (ESCs). Upon SUMOylation depletion, the chromatin becomes de-compacted and H1 is evicted, leading to totipotency reactivation. Furthermore, we show that H1 and SUMO2/3 jointly mediate the repression of totipotent elements. Lastly, we demonstrate that preventing SUMOylation on H1 abrogates its ability to repress the totipotency program in ESCs. Collectively, our findings unravel a critical role for SUMOylation of H1 in facilitating chromatin repression and desolation of the totipotent identity.

Maintenance of differentiated cellular states is crucial for numerous biological processes, yet its molecular basis remains unclear. Here, we investigate how mechanistically regulatory T (Treg) cell fate is “locked in” during lineage commitment via transcriptional regulation of its lineage-specifying factor Foxp3. Tet-mediated DNA demethylation ofFoxp3enhancer CNS2 was proposed to be a key mechanism maintaining Foxp3 transcription. However, this model has not been directly tested. Therefore, we integrated genetic, pharmacological, and epigenetic approaches to examine the function and mechanism of DNA demethylation in Treglineage maintenance. We observed an abrupt switch of the transcriptional drivers of Foxp3 upon DNA demethylation, which was abolished by CNS2 deficiency. Demethylation of CNS2 increased chromatin accessibility and protein binding, conferring on Tregfate substantial resistance to adverse environments. Thus, our study consolidated the role of DNA demethylation in stabilizing Foxp3 expression incisand revealed a novel regulatory mode governing Tregidentity.

Primordial germ cells (PGCs) are common ancestors of all germline cells. In mammals, PGCs emerge in early-stage embryos around the timing of gastrulation at or near epiblast, and specification of PGCs from their precursor cells involves multiple growth factors secreted by adjacent cells. Recent advancements in germline stem cell biology have made it possible to generate PGC-like cell culture models (PGCLCs for PGC-like cells) from human and mouse pluripotent stem cells by mimicking the embryonic growth factor environment in vitro. Here we describe a method of producing human PGCLCs from primed-pluripotency induced pluripotent stem cells (iPSCs) via temporal conversion to naive pluripotency followed by formation of embryoid bodies (EBs) using the spin-EB method.

Primordial germ cells (PGCs) are common ancestors of all germline cells. In mammals, PGCs emerge in early-stage embryos around the timing of gastrulation at or near epiblast, and specification of PGCs from their precursor cells involves multiple growth factors secreted by adjacent cells. Recent advancements in germline stem cell biology have made it possible to generate PGC-like cell culture models (PGCLCs for PGC-like cells) from human and mouse pluripotent stem cells by mimicking the embryonic growth factor environment in vitro. Here we describe a method of producing human PGCLCs from primed-pluripotency induced pluripotent stem cells (iPSCs) via temporal conversion to naive pluripotency followed by formation of embryoid bodies (EBs) using the spin-EB method.

SummaryEpileptic encephalopathies (EEs) are a group of disorders associated with intractable seizures, brain development and functional abnormalities, and in some cases, premature death. Pathogenic human germline biallelic mutations in tumor suppressor WW domain-containing oxidoreductase (WWOX) are associated with a relatively mild autosomal-recessive spinocerebellar ataxia-12 (SCAR12) and a more severe early infantile WWOX-related epileptic encephalopathy (WOREE). In this study, we generated an in-vitro model for EEs, using the devastating WOREE syndrome as a prototype, by establishing brain organoids from CRISPR-engineered human ES cells and from patient-derived iPSCs. Using these models, we discovered dramatic cellular and molecular CNS abnormalities, including neural population changes, cortical differentiation malfunctions, and Wnt-pathway and DNA-damage response impairment. Furthermore, we provide a proof-of-concept that ectopic WWOX expression could potentially rescue these phenotypes. Our findings underscore the utility of modeling childhood epileptic encephalopathies using brain organoids and their use as a unique platform to test possible therapeutic intervention strategies.

The mammalian body plan is established shortly after the embryo implants into the maternal uterus, and our understanding of post-implantation developmental processes remains limited. Although pre- and peri-implantation mouse embryos are routinely cultured in vitro1,2, approaches for the robust culture of post-implantation embryos from egg cylinder stages until advanced organogenesis remain to be established. Here we present highly effective platforms for the ex utero culture of post-implantation mouse embryos, which enable the appropriate development of embryos from before gastrulation (embryonic day (E) 5.5) until the hindlimb formation stage (E11). Late gastrulating embryos (E7.5) are grown in three-dimensional rotating bottles, whereas extended culture from pre-gastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle culture platforms. Histological, molecular and single-cell RNA sequencing analyses confirm that the ex utero cultured embryos recapitulate in utero development precisely. This culture system is amenable to the introduction of a variety of embryonic perturbations and micro-manipulations, the results of which can be followed ex utero for up to six days. The establishment of a system for robustly growing normal mouse embryos ex utero from pre-gastrulation to advanced organogenesis represents a valuable tool for investigating embryogenesis, as it eliminates the uterine barrier and allows researchers to mechanistically interrogate post-implantation morphogenesis and artificial embryogenesis in mammals. A new culture system makes it possible to grow mouse embryos and study their development outside the uterus up to the point of late organogenesis.

Summary Isolating human MEK/ERK signaling-independent pluripotent stem cells (PSCs) with naive pluripotency characteristics while maintaining differentiation competence and (epi)genetic integrity remains challenging. Here, we engineer reporter systems that allow the screening for defined conditions that induce molecular and functional features of human naive pluripotency. Synergistic inhibition of WNT/β-CATENIN, protein kinase C (PKC), and SRC signaling consolidates the induction of teratoma-competent naive human PSCs, with the capacity to differentiate into trophoblast stem cells (TSCs) and extraembryonic naive endodermal (nEND) cells in vitro. Divergent signaling and transcriptional requirements for boosting naive pluripotency were found between mouse and human. P53 depletion in naive hPSCs increased their contribution to mouse-human cross-species chimeric embryos upon priming and differentiation. Finally, MEK/ERK inhibition can be substituted with the inhibition of NOTCH/RBPj, which induces alternative naive-like hPSCs with a diminished risk for deleterious global DNA hypomethylation. Our findings set a framework for defining the signaling foundations of human naive pluripotency., Graphical abstract, Highlights • Inhibition of SRC, PKC, and WNT consolidates human naive pluripotency induction • Competitiveness of p53 depleted human PSCs in cross-species chimeric embryos • Opposing net effect for ACTIVIN and WNT on mouse versus human naive pluripotency • 2i and ERKi independent alternative human naive-like PSC conditions, Engineered systems were used to screen for conditions that enable robust induction of human naive PSCs without the obligation for exogenous transgenes or feeder cells. The latter allowed defining the signaling and transcriptional foundations of human naive PSCs with enhanced (epi)genetic stability and competence for differentiation into all lineages.

Different conditions have been devised to isolate MEK/ERK signalling independent human naïve pluripotent stem cells (PSCs) that are distinct from conventional primed PSCs and better correspond to pre-implantation developmental stages. While the naïve conditions described thus far endow human PSCs with different extents of naivety features, isolating human pluripotent cells that retain characteristics of ground state pluripotency while maintaining differentiation potential and genetic integrity, remains a major challenge. Here we engineer reporter systems that allow functional screening for conditions that can endow both the molecular and functional features expected from human naive pluripotency. We establish that simultaneous inhibition of SRC-NFκB, WNT/ßCATENIN and PKC signalling pathways is essential for enabling expansion of teratoma competent fully naïve human PSCs in defined or xeno-free conditions. Divergent signalling and transcriptional requirements for maintaining naïve pluripotency were found between mouse and human. Finally, we establish alternative naïve conditions in which MEK/ERK inhibition is substituted with inhibition for NOTCH/RBPj signalling, which allow obtaining alternative human naïve PSCs with diminished risk for loss of imprinting and deleterious global DNA hypomethylation. Our findings set a framework for the signalling foundations of human naïve pluripotency and may advance its utilization in future translational applications.Highlights of key findingsCombined inhibition of SRC, WNT and PKC signaling consolidates human naïve pluripotencyStable expansion of DNA/RNA methylation-independent and TGF/ACTIVIN-independent human naïve PSCsOpposing roles for ACTIVIN and WNT/ßCATENIN signaling on mouse vs. human naive pluripotency2i and MEK/ERKi independent alternative human naïve PSC conditions via inhibiting NOTCH/RBPj signaling

The N6-methyladenosine (m(6)A) modification is the most prevalent post-transcriptional mRNA modification, regulating mRNA decay and splicing. It plays a major role during normal development, differentiation, and disease progression. The modification is regulated by a set of writer, eraser, and reader proteins. The YTH domain family of proteins consists of three homologous m(6)A-binding proteins, Ythdf1, Ythdf2, and Ythdf3, which were suggested to have different cellular functions. However, their sequence similarity and their tendency to bind the same targets suggest that they may have overlapping roles. We systematically knocked out (KO) the Mettl3 writer, each of the Ythdf readers, and the three readers together (triple-KO). We then estimated the effect in vivo in mouse gametogenesis, postnatal viability, and in vitro in mouse embryonic stem cells (mESCs). In gametogenesis, Mettl3-KO severity is increased as the deletion occurs earlier in the process, and Ythdf2 has a dominant role that cannot be compensated by Ythdf1 or Ythdf3, due to differences in readers’ expression pattern across different cell types, both in quantity and in spatial location. Knocking out the three readers together and systematically testing viable offspring genotypes revealed a redundancy in the readers’ role during early development that is Ythdf1/2/3 gene dosage-dependent. Finally, in mESCs there is compensation between the three Ythdf reader proteins, since the resistance to differentiate and the significant effect on mRNA decay occur only in the triple-KO cells and not in the single KOs. Thus, we suggest a new model for the Ythdf readers function, in which there is profound dosage-dependent redundancy when all three readers are equivalently coexpressed in the same cell types.

Human pluripotent stem cells (hPSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have a well-orchestrated program for differentiation and self-renewal. However, the structural features of unique proteostatic-maintaining mechanisms in hPSCs and their features, distinct from those of differentiated cells, in response to cellular stress remain unclear. We evaluated and compared the morphological features and stress response of hPSCs and fibroblasts. Compared to fibroblasts, electron microscopy showed simpler/fewer structures with fewer networks in the endoplasmic reticulum (ER) of hPSCs, as well as lower expression of ER-related genes according to meta-analysis. As hPSCs contain low levels of binding immunoglobulin protein (BiP), an ER chaperone, thapsigargin treatment sharply increased the gene expression of the unfolded protein response. Thus, hPSCs with decreased chaperone function reacted sensitively to ER stress and entered apoptosis faster than fibroblasts. Such ER stress-induced apoptotic processes were abolished by tauroursodeoxycholic acid, an ER-stress reliever. Hence, our results revealed that as PSCs have an underdeveloped structure and express fewer BiP chaperone proteins than somatic cells, they are more susceptible to ER stress-induced apoptosis in response to stress.

The rising field of RNA modifications is stimulating massive research nowadays. m6A, the most abundant mRNA modification is highly conserved during evolution. Through the last decade, the essential components of this dynamic mRNA modification machinery were found and classified into writer, eraser and reader proteins. m6A modification is now known to take part in diverse biological processes such as embryonic development, cell circadian rhythms and cancer stem cell proliferation. In addition, there is already firm evidence for the importance of m6A modification in stem cell differentiation and gametogenesis, both in males and females. This review attempts to summarize the important results of recent years studying the mechanism underlying stem cell differentiation and gametogenesis processes.

The rising field of RNA modifications is stimulating massive research nowadays. m6A, the most abundant mRNA modification is highly conserved during evolution. Through the last decade, the essential components of this dynamic mRNA modification machinery were found and classified into writer, eraser and reader proteins. m6A modification is now known to take part in diverse biological processes such as embryonic development, cell circadian rhythms and cancer stem cell proliferation. In addition, there is already firm evidence for the importance of m6A modification in stem cell differentiation and gametogenesis, both in males and females. This review attempts to summarize the important results of recent years studying the mechanism underlying stem cell differentiation and gametogenesis processes.

β-Catenin recruits BRD4 and other coregulators to protect pluripotency gene transcription against network perturbation., Mouse embryonic stem cells cultured with MEK (mitogen-activated protein kinase kinase) and GSK3 (glycogen synthase kinase 3) inhibitors (2i) more closely resemble the inner cell mass of preimplantation blastocysts than those cultured with SL [serum/leukemia inhibitory factor (LIF)]. The transcriptional mechanisms governing this pluripotent ground state are unresolved. Release of promoter-proximal paused RNA polymerase II (Pol2) is a multistep process necessary for pluripotency and cell cycle gene transcription in SL. We show that β-catenin, stabilized by GSK3 inhibition in medium with 2i, supplies transcriptional coregulators at pluripotency loci. This selectively strengthens pluripotency loci and renders them addicted to transcription initiation for productive gene body elongation in detriment to Pol2 pause release. By contrast, cell cycle genes are not bound by β-catenin, and proliferation/self-renewal remains tightly controlled by Pol2 pause release under 2i conditions. Our findings explain how pluripotency is reinforced in the ground state and also provide a general model for transcriptional resilience/adaptation upon network perturbation in other contexts.

Nuclear factor Foxp3 sustains immune-suppressive function of regulatory T (Treg) cells during their long lifespan, raising a fundamental question about how stochastic Foxp3 induction is transformed into stable Foxp3 expression during Treg lineage commitment. To address this issue, we examined the processes of Treg development and observed an abrupt switch of the transcriptional mechanisms regulating Foxp3 expression. By integrating genetic and pharmacological approaches with unbiased CRISPR screening, we discovered several known and novel nuclear factors spanning a diverse aspects of gene regulation that control Foxp3 induction through a super-additive effect. After Tet-dependent DNA demethylation, Foxp3 transcription was governed by a robust mechanism involving redundant nuclear programs and a shift of Foxp3 enhancers. These distinct regulatory modes transform stochastic induction to a stable state of Foxp3 transcription. Collectively, our results revealed novel regulators and redefined the contrasting mechanisms governing Foxp3 transcription for Treg selection and lineage maintenance.

The N6-methyladenosine (m6A) modification is the most prevalent post-transcriptional mRNA modification, regulating mRNA decay and splicing. It plays a major role during normal development, differentiation, and disease progression. The modification is regulated by a set of writer, eraser, and reader proteins. The YTH domain family of proteins consists of three homologous m6A-binding proteins, Ythdf1, Ythdf2, and Ythdf3, which were suggested to have different cellular functions. However, their sequence similarity and their tendency to bind the same targets suggest that they may have overlapping roles. We systematically knocked out (KO) the Mettl3 writer, each of the Ythdf readers, and the three readers together (triple-KO). We then estimated the effect in vivo in mouse gametogenesis, postnatal viability, and in vitro in mouse embryonic stem cells (mESCs). In gametogenesis, Mettl3-KO severity is increased as the deletion occurs earlier in the process, and Ythdf2 has a dominant role that cannot be compensated by Ythdf1 or Ythdf3, due to differences in readers’ expression pattern across different cell types, both in quantity and in spatial location. Knocking out the three readers together and systematically testing viable offspring genotypes revealed a redundancy in the readers’ role during early development that is Ythdf1/2/3 gene dosage-dependent. Finally, in mESCs there is compensation between the three Ythdf reader proteins, since the resistance to differentiate and the significant effect on mRNA decay occur only in the triple-KO cells and not in the single KOs. Thus, we suggest a new model for the Ythdf readers function, in which there is profound dosage-dependent redundancy when all three readers are equivalently coexpressed in the same cell types.

Summary 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., Graphical Abstract, Highlights • RAS is activated upon early differentiation of mouse embryonic stem cells (mESCs) • RAS repression by LIF prevents early differentiation • RAS regulates the transition from naive to primed state of mESCs • RAS regulates glycolysis, chromatin remodeling, and CADHERIN expression, Altshuler et al. report that RAS activation positively regulated key processes of naive-primed transition of mouse embryonic stem cells, including changes in metabolism, chromatin remodeling, and the switch in CADHERIN expression. Pharmacological inhibition of RAS attenuated cellular priming, suggesting that RAS inhibition may be potentially useful for converting human cells into ground state and for efficient somatic cellular reprogramming.

Human brain wrinkling has been implicated in neurodevelopmental disorders and yet its origins remain unknown. Polymer gel models suggest that wrinkling emerges spontaneously due to compression forces arising during differential swelling, but these ideas have not been tested in a living system. Here, we report the appearance of surface wrinkles during the in vitro development and self-organization of human brain organoids in a microfabricated compartment that supports in situ imaging over a timescale of weeks. We observe the emergence of convolutions at a critical cell density and maximal nuclear strain, which are indicative of a mechanical instability. We identify two opposing forces contributing to differential growth: cytoskeletal contraction at the organoid core and cell-cycle-dependent nuclear expansion at the organoid perimeter. The wrinkling wavelength exhibits linear scaling with tissue thickness, consistent with balanced bending and stretching energies. Lissencephalic (smooth brain) organoids display reduced convolutions, modified scaling and a reduced elastic modulus. Although the mechanism here does not include the neuronal migration seen in vivo, it models the physics of the folding brain remarkably well. Our on-chip approach offers a means for studying the emergent properties of organoid development, with implications for the embryonic human brain. Wrinkling in human brain organoids suggests that brain development may be mechanically driven, a notion supported only by model gels so far. Evidence in this simple living system highlights roles for cytoskeletal contraction and nuclear expansion.

The implications of scientific breakthroughs are rarely faced up to in advance of their realization. Stem cell-derived human gametes, a disruptive technology in waiting, are likely to recapitulate this historic pattern absent active intervention. Herein we call for the conduct of thoughtful ante hoc deliberations on the prospect of stem cell-derived human gametes with an eye toward minimizing potential untoward post hoc regulatory or statutory impositions.

Immunoglobulin heavy chain (IgH) locus-associated G-rich long noncoding RNA (SμGLT) is important for physiological and pathological B cell DNA recombination. We demonstrate that the METTL3 enzyme-catalyzed N6-methyladenosine (m6A) RNA modification drives recognition and 3' end processing of SμGLT by the RNA exosome, promoting class switch recombination (CSR) and suppressing chromosomal translocations. The recognition is driven by interaction of the MPP6 adaptor protein with nuclear m6A reader YTHDC1. MPP6 and YTHDC1 promote CSR by recruiting AID and the RNA exosome to actively transcribe SμGLT. Direct suppression of m6A modification of SμGLT or of m6A reader YTHDC1 reduces CSR. Moreover, METTL3, an essential gene for B cell development in the bone marrow and germinal center, suppresses IgH-associated aberrant DNA breaks and prevents genomic instability. Taken together, we propose coordinated and central roles for MPP6, m6A modification, and m6A reader proteins in controlling long noncoding RNA processing, DNA recombination, and development in B cells.

Natural Killer (NK) cells employ activating receptors like the Natural Cytotoxicity Receptors (NCRs: NKp30, NKp44 and NKp46), of which only NKp46 has a mouse orthologue (Ncr1), to eliminate abnormal cells. NKp46/Ncr1 is considered a selective marker for NK cells, although it is also found on a subset of ILCs, where it appears to be without function. The influenza virus hemagglutinin (HA) was the first ligand identified for Ncr1/NKp46 followed by other viral, bacterial and even fungal ligands. NKp46/Ncr1 also recognizes unknown self and tumor ligands. Here we describe the generation of a transgenic mouse where the Ncr1 gene is expressed in the Rosa locus, preceded by a floxed stop sequence allowing Ncr1/NKp46 expression in various tissues upon crossing with Cre transgenic mouse lines. Surprisingly, while several crossings were attempted, Ncr1 overexpression was successful only where cre recombinase expression was dependent on the Ncr1 promoter. Ncr1 overexpression in NK cells increased NK cell immunity in two hallmark Ncr1 related pathologies, influenza virus infection and B16 melanoma. These data suggest that increasing NK cell cytotoxicity by enforced NKp46/Ncr1 expression serves as a potential therapeutic opportunity for the treatment of various pathologies, and in immunotherapy.

Immunoglobulin heavy chain (IgH) locus-associated G-rich long noncoding RNA (SμGLT) is important for physiological and pathological B cell DNA recombination. We demonstrate that the METTL3 enzyme-catalyzed N(6)-methyladenosine (m(6)A) RNA modification drives recognition and 3’-end processing of SμGLT by the RNA exosome, promoting class switch recombination (CSR) and suppressing chromosomal translocations. The recognition is driven by interaction of the MPP6 adaptor protein with nuclear m(6)A reader YTHDC1. MPP6 and YTHDC1 promote CSR by recruiting AID and the RNA exosome to actively transcribing SμGLT. Direct suppression of m(6)A modification of SμGLT or of m(6)A reader YTHDC1 reduces CSR. Moreover, METTL3, an essential gene for B cell development in the bone marrow and germinal center, suppresses IgHassociated aberrant DNA breaks and prevents genomic instability. Taken together, we propose coordinated and central roles for MPP6, m(6)A modification, and m(6)A reader proteins in controlling long noncoding RNA processing, DNA recombination and development in B cells.

N6-methyladenosine (m6A) is the most abundant modification on mRNA, and is implicated in critical roles in development, physiology and disease. The ability to map m6A using immunoprecipitation-based approaches has played a critical role in dissecting m6A functions and mechanisms of action. Yet, these approaches are of limited specificity, unknown sensitivity, and unable to quantify m6A stoichiometry. These limitations have severely hampered our ability to unravel the factors determining where m6A will be deposited, to which levels (the ‘m6A code’), and to quantitatively profile m6A dynamics across biological systems. Here, we used the RNase MazF, which cleaves specifically at unmethylated RNA sites, to develop MASTER-seq for systematic quantitative profiling of m6A sites at 16-25% of all m6A sites at single nucleotide resolution. We established MASTER-seq for orthogonal validation andde novodetection of m6A sites, and for tracking of m6A dynamics in yeast gametogenesis and in early mammalian differentiation. We discover that antibody-based approaches severely underestimate the number of m6A sites, and that both the presence of m6A and its stoichiometry are ‘hard-coded’ via a simple and predictable code within the extended sequence composition at the methylation sites. This code accounts for ~50% of the variability in methylation levels across sites, allows excellentde novoprediction of methylation sites, and predicts methylation acquisition and loss across evolution. We anticipate that MASTER-seq will pave the path towards a more quantitative investigation of m6A biogenesis and regulation in a wide variety of systems, including diverse cell types, stimuli, subcellular components, and disease states.

Primordial germ cells (PGCs) are common precursors of all germline cells. In mouse embryos, a founding population of ~40 PGCs are induced from pluripotent epiblast cells by orchestrated exposures to cytokines, including bone morphogenetic protein 4 (Bmp4). In human embryos, the earliest PGCs have been identified on the endodermal wall of yolk sac around the end of the 3rd week of gestation, but little is known about the process of human PGC specification and their early development. To circumvent the technical and ethical barriers of studying human embryonic PGCs, surrogate cell culture models have been recently generated from pluripotent stem cells. Here, we describe a 13-day protocol for robust production of human PGC-Like Cells (hPGCLCs). Human induced pluripotent stem cells (hiPSCs) maintained in the primed pluripotency state are incubated in the 4i naive reprogramming medium for 48 hours, dissociated to single cells, and packed into microwells. Prolonged maintenance of hiPSCs in the naive pluripotency state causes significant chromosomal aberrations and should be avoided. hiPSCs in the microwells are maintained for an additional 24 hours in the 4i medium to form embryoid bodies (EBs), which are then cultured in low-adherence plasticware under a rocking condition in the hPGCLC induction medium containing a high concentration of recombinant human BMP4. EBs are further cultured for up to 8 days in the rocking, non-adherent condition to obtain maximum yields of hPGCLCs. By immunohistochemistry, hPGCLCs are readily detected as cells strongly expressing OCT4 in almost all EBs exclusively on their surface. When EBs are enzymatically dissociated and subjected to FACS enrichment, hPGCLCs can be collected as CD38+ cells with up to 40-45% yield.

In the version of this article initially published, the penultimate sentence of the abstract included a typographical error ('cxgenes'). The correct word is 'genes'. The error has been corrected in the HTML and PDF version of the article.

Histone modifications play an important role in chromatin organization and transcriptional regulation, but despite the large amount of genome-wide histone modification data collected in different cells and tissues, little is known about co-occurrence of modifications on the same nucleosome. Here we present a genome-wide quantitative method for combinatorial indexed chromatin immunoprecipitation (co-ChIP) to characterize co-occurrence of histone modifications on nucleosomes. Using co-ChIP, we study the genome-wide co-occurrence of 14 chromatin marks (70 pairwise combinations), and find previously undescribed co-occurrence patterns, including the co-occurrence of H3K9me1 and H3K27ac in super-enhancers. Finally, we apply co-ChIP to measure the distribution of the bivalent H3K4me3-H3K27me3 domains in two distinct mouse embryonic stem cell (mESC) states and in four adult tissues. We observe dynamic changes in 5,786 regions and discover both loss and de novo gain of bivalency in key tissue-specific regulatory genes, suggesting a functional role for bivalent domains during different stages of development. These results show that co-ChIP can reveal the complex interactions between histone modifications.