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Abstract Background Pluripotent states of embryonic stem cells (ESCs) with distinct transcriptional profiles affect ESC differentiative capacity and therapeutic potential. Although single-cell RNA sequencing has revealed additional subpopulations and specific features of naive and primed human pluripotent stem cells (hPSCs), the underlying mechanisms that regulate their specific transcription and that control their pluripotent states remain elusive. Results By single-cell analysis of high-resolution, three-dimensional (3D) genomic structure, we herein demonstrate that remodeling of genomic structure is highly associated with the pluripotent states of human ESCs (hESCs). The naive pluripotent state is featured with specialized 3D genomic structures and clear chromatin compartmentalization that is distinct from the primed state. The naive pluripotent state is achieved by remodeling the active euchromatin compartment and reducing chromatin interactions at the nuclear center. This unique genomic organization is linked to enhanced chromatin accessibility on enhancers and elevated expression levels of naive pluripotent genes localized to this region. In contradistinction, the primed state exhibits intermingled genomic organization. Moreover, active euchromatin and primed pluripotent genes are distributed at the nuclear periphery, while repressive heterochromatin is densely concentrated at the nuclear center, reducing chromatin accessibility and the transcription of naive genes. Conclusions Our data provide insights into the chromatin structure of ESCs in their naive and primed states, and we identify specific patterns of modifications in transcription and chromatin structure that might explain the genes that are differentially expressed between naive and primed hESCs. Thus, the inversion or relocation of heterochromatin to euchromatin via compartmentalization is related to the regulation of chromatin accessibility, thereby defining pluripotent states and cellular identity.

Embryonic development is a continuum in vivo. Transcriptional analysis can separate established human embryonic stem cells (hESC) into at least four distinct developmental pluripotent stages, two naïve and two primed, early and late relative to the intact epiblast. In this study we primarily show that exposure of frozen human blastocysts to an inhibitor of checkpoint kinase 1 (CHK1) upon thaw greatly enhances establishment of karyotypically normal late naïve hESC cultures. These late naïve cells are plastic and can be toggled back to early naïve and forward to early primed pluripotent stages. The early primed cells are transcriptionally equivalent to the post inner cell mass intermediate (PICMI) stage seen one day following transfer of human blastocysts into in vitro culture and are stable at an earlier stage than conventional primed hESC. [ABSTRACT FROM AUTHOR]

Inhibition of PKC (PKCi) signaling maintains pluripotency of embryonic stem cells (ESCs) across different mammalian species. However, the position of PKCi maintained ESCs in the pluripotency continuum is largely unknown. Here we demonstrate that mouse ESCs when cultured continuously, with PKCi, for 75 days are retained in naïve state of pluripotency. Gene expression analysis and proteomics studies demonstrated enhanced naïve character of PKCi maintained ESCs in comparison to classical serum/LIF (S/L) supported ESCs. Molecular analysis revealed that activation of PKCζ isoform associate with primed state of pluripotency, present in epiblast-like stem cells generated in vitro while inhibition of PKCζ phosphorylation associated with naïve state of pluripotency in vitro and in vivo. Phosphoproteomics and chromatin modification enzyme array based studies showed loss in DNA methyl transferase 3B (DNMT3B) and its phosphorylation level upon functional inhibition of PKCζ as one of the crucial components of this regulatory pathway. Unlike ground state of pluripotency maintained by MEK/GSK3 inhibitor in addition to LIF (2i/LIF), loss in DNMT3B is a reversible phenomenon in PKCi maintained ESCs. Absence of phosphorylation of c-MYC, RAF1, SPRY4 while presence of ERF, DUSP6, CIC and YAP1 phosphorylation underlined the phosphoproteomics signature of PKCi mediated maintenance of naïve pluripotency. States of pluripotency represent the developmental continuum and the existence of PKCi mediated mouse ESCs in a distinct state in the continuum of pluripotency (DiSCo) might contribute to the establishment of stages of murine embryonic development that were non-permissible till date. [ABSTRACT FROM AUTHOR]

Human pluripotent stem cells provide an inexhaustible model to study human embryogenesis in vitro. Recent studies have provided diverse models to generate human blastoids by self-organization of different pluripotent stem cells or somatic reprogramming intermediates. However, whether blastoids can be generated from other cell types or whether they can recapitulate postimplantation development in vitro is unknown. Here, we develop a strategy to generate human blastoids from heterogeneous intermediates with epiblast, trophectoderm, and primitive endoderm signatures of the primed-to-naïve conversion process, which resemble natural blastocysts in morphological architecture, composition of cell lineages, transcriptome, and lineage differentiation potential. In addition, these blastoids reflect many features of human peri-implantation and pregastrulation development when further cultured in an in vitro 3D culture system. In summary, our study provides an alternative strategy to generate human blastoids and offers insights into human early embryogenesis by modeling peri- and postimplantation development in vitro. [ABSTRACT FROM AUTHOR]

The therapeutic potential of mesenchymal stem/stromal cells (MSCs) is limited by acquired senescence following prolonged culture expansion and high-passage numbers. However, the degree of cell senescence is dynamic, and cell-cell communication is critical to promote cell survival. MSC spheroids exhibit improved viability compared with monodispersed cells, and actin-rich tunneling nanotubes (TNTs) may mediate cell survival and other functions through the exchange of cytoplasmic components. Building upon our previous demonstration of TNTs bridging MSCs within these cell aggregates, we hypothesized that TNTs would influence the expression of senescence markers in MSC spheroids. We confirmed the existence of functional TNTs in MSC spheroids formed from low-passage, high-passage, and mixtures of low- and high-passage cells using scanning electron microscopy, confocal microscopy, and flow cytometry. The contribution of TNTs toward the expression of senescence markers was investigated by blocking TNT formation with cytochalasin D (CytoD), an inhibitor of actin polymerization. CytoD-treated spheroids exhibited decreases in cytosol transfer. Compared with spheroids formed solely of high-passage MSCs, the addition of low-passage MSCs reduced p16 expression, a known genetic marker of senescence. We observed a significant increase in p16 expression in high-passage cells when TNT formation was inhibited, establishing the importance of TNTs in MSC spheroids. These data confirm the restorative role of TNTs within MSC spheroids formed with low- and high-passage cells and represent an exciting approach to use higher-passage cells in cell-based therapies.

Pluripotent character is described as the potency of cells to differentiate into all three germ layers. The best example to reinstate the term lies in the context of embryonic stem cells (ESCs). Pluripotent ESC describes the in vitro status of those cells that originate during the complex process of embryogenesis. Pre-implantation to post-implantation development of embryo embrace cells with different levels of stemness. Currently, four states of pluripotency have been recognized, in the progressing order of "naïve," "poised," "formative," and "primed." Epigenetics act as the "conductor" in this "orchestra" of transition in pluripotent states. With a distinguishable gene expression profile, these four states associate with different epigenetic signatures, sometimes distinct while otherwise overlapping. The present review focuses on how epigenetic factors, including DNA methylation, bivalent chromatin, chromatin remodelers, chromatin/nuclear architecture, and microRNA, could dictate pluripotent states and their transition among themselves. [ABSTRACT FROM AUTHOR]

Background: A MenABCWY vaccine containing 4CMenB and MenACWY-CRM vaccine components has been developed to protect against the five meningococcal serogroups that cause most invasive disease cases., Methods: In this phase 3 study (NCT04707391), healthy participants aged 15-25 years, who had received MenACWY vaccination ≥4 years previously, were randomized (1:1) to receive two MenABCWY doses six months apart or one MenACWY-CRM dose. Primary objectives were to demonstrate the non-inferiority of MenABCWY 1 month post-vaccination versus MenACWY-CRM, with a lower limit of 2-sided 95% confidence interval above -10% for group differences in 4-fold rise in human serum bactericidal antibody (hSBA) titers against serogroups ACWY, and to evaluate reactogenicity and safety. Secondary endpoints included percentages of participants with hSBA titers ≥lower limit of quantitation (LLOQ) against serogroups ACWY and vaccine antigen-specific serogroup B (MenB) indicator strains., Results: Non-inferiority of MenABCWY versus MenACWY-CRM was demonstrated following each MenABCWY dose. Percentages of participants with hSBA titers ≥LLOQ for serogroups ACWY were 97.9-98.9% and 99.5-100% following one and two MenABCWY doses, respectively, and 96.8-99.0% following one MenACWY-CRM dose. After two MenABCWY doses, 75.6-96.3% of participants had hSBA titers ≥LLOQ against MenB indicator strains. The MenABCWY vaccine was well tolerated in MenACWY-primed individuals with a favorable safety profile., Conclusions: Immune responses against serogroups ACWY following one and two doses of investigational MenABCWY vaccine are non-inferior to those following MenACWY-CRM in MenACWY-primed adolescents and young adults. Robust immune responses were observed against MenB indicator strains after two MenABCWY doses administered 6 months apart., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Pluripotent stem cells could self-renew and differentiate into various cells. In particular, porcine pluripotent stem cells are useful for preclinical therapy, transgenic animals, and agricultural usage. These stem cells have naïve and primed pluripotent states. Naïve pluripotent stem cells represented by mouse embryonic stem cells form chimeras after blastocyst injection. Primed pluripotent stem cells represented by mouse epiblast stem cells and human embryonic stem cells. They could not produce chimeras after blastocyst injection. Populations of embryonic stem cells are not homogenous; therefore, reporter systems are used to clarify the status of stem cells and to isolate the cells. For this reason, studies of the OCT4 reporter system have been conducted for decades. This review will discuss the naïve and primed pluripotent states and recent progress in the development of porcine OCT4 reporter systems.

Pluripotent cells in mouse embryos, which first emerge in the inner cell mass of the blastocyst, undergo gradual transition marked by changes in gene expression, developmental potential, polarity, and morphology as they develop from the pre‐implantation until post‐implantation gastrula stage. Recent studies of cultured mouse pluripotent stem cells (PSCs) have clarified the presence of intermediate pluripotent stages between the naïve pluripotent state represented by embryonic stem cells (ESCs—equivalent to the pre‐implantation epiblast) and the primed pluripotent state represented by epiblast stem cells (EpiSCs—equivalent to the late post‐implantation gastrula epiblast). In this review, we discuss these recent findings in light of our knowledge on peri‐implantation mouse development and consider the implications of these new PSCs to understand their temporal sequence and the feasibility of using them as model system for pluripotency. [ABSTRACT FROM AUTHOR]

Pluripotent cells have been stabilized from pre- and post-implantation blastocysts, representing respectively naïve and primed stages of embryonic stem cells (ESCs) with distinct epigenetic, metabolic and transcriptomic features. Beside these two well characterized pluripotent stages, several intermediate states have been reported, as well as a small subpopulation of cells that have reacquired features of the 2C-embryo (2C-like cells) in naïve mouse ESC culture. Altogether, these represent a continuum of distinct pluripotency stages, characterized by metabolic transitions, for which we propose a new role for a long-known metabolite: succinate. Mostly seen as the metabolite of the TCA, succinate is also at the crossroad of several mitochondrial biochemical pathways. Its role also extends far beyond the mitochondrion, as it can be secreted, modify proteins by lysine succinylation and inhibit the activity of alpha-ketoglutarate-dependent dioxygenases, such as prolyl hydroxylase (PHDs) or histone and DNA demethylases. When released in the extracellular compartment, succinate can trigger several key transduction pathways after binding to SUCNR1, a G-Protein Coupled Receptor. In this review, we highlight the different intra- and extracellular roles that succinate might play in the fields of early pluripotency and embryo development. [ABSTRACT FROM AUTHOR]

Cellular metabolism plays both an active and passive role in embryonic development, pluripotency, and cell-fate decisions. However, little is known regarding the role of metabolism in regulating the recently described "formative" pluripotent state. The pluripotent developmental continuum features a metabolic switch from a bivalent metabolism (both glycolysis and oxidative phosphorylation) in naive cells, to predominantly glycolysis in primed cells. We investigated the role of pyruvate kinase muscle isoforms 1/2 (PKM1/2) in naive, formative, and primed mouse embryonic stem cells through modulation of PKM1/2 messenger RNA transcripts using steric blocking morpholinos that downregulate PKM2 and upregulate PKM1. We have examined these effects in naive, formative, and primed cells by quantifying the effects of PKM1/2 modulation on pluripotent and metabolic transcripts and by measuring shifts in the population frequencies of cells expressing naive and primed cell surface markers by flow cytometry. Our results demonstrate that modulating PKM1 and PKM2 levels alters the transition from the naive state into a primed pluripotent state by enhancing the proportion of the affected cells seen in the "formative" state. Therefore, we conclude that PKM1/2 actively contributes to mechanisms that oversee early stem pluripotency and their progression toward a primed pluripotent state. [ABSTRACT FROM AUTHOR]

Conventional human pluripotent stem cells (hPSCs), known for being in a primed state, are pivotal for both basic research and clinical applications since such cells produce various types of differentiated cells. Recent reports on PSCs shed light on the pluripotent hierarchy of stem cells and have promoted the exploration of new stem cell states along with their culture systems. Human naïve PSCs are expected to provide further knowledge of early developmental mechanisms and improvements for differentiation programmes in the regenerative therapy of conventionally primed PSCs. However, practical challenges exist in using naïve-state PSCs such as determining the conditions for hypoxic culture condition and showing limited stable cellular proliferation. Here, we have developed new leukemia inhibitory factor dependent PSCs by applying our previous work, the combination of dibenzazepine and a DOT1L inhibitor to achieve the stable culture of naïve-state PSCs. The potential of these cells to differentiate into all three germ layers was shown both in vitro and in vivo. Such new naïve-state PSCs formed dome-shaped colonies at a faster rate than conventional, primed-state human induced PSCs and could be maintained for an extended period in the absence of hypoxic culture conditions. We also identified relatively high expression levels of naïve cell markers. Thus, non-hypoxia treated, leukemia inhibitory factor-dependent PSCs are anticipated to have characteristics similar to those of naïve-like PSCs, and to enhance the utility value of PSCs. Such naïve PSCs may allow the molecular characterization of previously undefined naïve human PSCs, and to ultimately contribute to the use of human pluripotent stem cells in regenerative medicine and disease modelling.

Human embryonic stem cells (hESCs) derived from blastocyst stage embryos present a primed state of pluripotency, whereas mouse ESCs (mESCs) display naïve pluripotency. Their unique characteristics make naïve hESCs more suitable for particular applications in biomedical research. This work aimed to derive hESCs from single blastomeres and determine their pluripotency state, which is currently unclear. We derived hESC lines from single blastomeres of 8-cell embryos and from whole blastocysts, and analysed several naïve pluripotency indicators, their transcriptomic profile and their trilineage differentiation potential. No significant differences were observed between blastomere-derived hESCs (bm-hESCs) and blastocyst-derived hESCs (bc-hESCs) for most naïve pluripotency indicators, including TFE3 localization, mitochondrial activity, and global DNA methylation and hydroxymethylation, nor for their trilineage differentiation potential. Nevertheless, bm-hESCs showed an increased single-cell clonogenicity and a higher expression of naïve pluripotency markers at early passages than bc-hESCs. Furthermore, RNA-seq revealed that bc-hESCs overexpressed a set of genes related to the post-implantational epiblast. Altogether, these results suggest that bm-hESCs, although displaying primed pluripotency, would be slightly closer to the naïve end of the pluripotency continuum than bc-hESCs., Competing Interests: Declaration of competing interest No competing interests declared., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Pluripotent stem cells (PSCs) are in vitro adaptations of in vivo pluripotency continuum and can be broadly classified into naïve state characteristic of pre-implantation epiblast and primed state resembling peri-gastrulation epiblasts. Naïve and primed PSCs differ in their cellular and molecular characteristics, e.g., molecular mechanisms for maintaining undifferentiated state. Naïve-to-primed PSC transition provides a tractable in vitro model to study pluripotency development in vivo. We previously developed a protocol that enabled high-efficient (100%) and homogenous derivation of ground state of primed epiblast stem cells (rsEpiSCs) by culturing the isolated post-implantation mouse epiblast under the culture condition containing FGF2 and a Wnt signaling inhibitor (IWR1) (F/R1 condition). Based on F/R1 condition, in this study, we developed three naïve-to-primed conversion methods for generating rsEpiSCs from naïve ground state of mouse ESCs (2i/LIF condition). We found that stepwise methods, but not directly, were effective for bona fide rsEpiSCs conversion from mouse ESCs. In sum, we established a robust and efficient ground states of naïve-to-primed PSC conversion strategy that will facilitate the study of genetic, epigenetic and metabolic processes involved in pluripotency progression in vivo. • A robust method of stepwise conversion from ground state-naïve ESCs to -primed pluripotent stem cells in mice. • p53 -mediated apoptosis accounts for the widespread cell death during the state transition. • "An epigenetic barrier" functions as a septum between naïve and primed states, which are gradually erased by the stepwise state transition. [ABSTRACT FROM AUTHOR]

Neuroinflammation contributes to Alzheimer's disease (AD) progression. Secondary inflammatory insults trigger delirium and can accelerate cognitive decline. Individual cellular contributors to this vulnerability require elucidation. Using APP/PS1 mice and AD brain, we studied secondary inflammatory insults to investigate hypersensitive responses in microglia, astrocytes, neurons, and human brain tissue. The NLRP3 inflammasome was assembled surrounding amyloid beta, and microglia were primed, facilitating exaggerated interleukin‐1β (IL‐1β) responses to subsequent LPS stimulation. Astrocytes were primed to produce exaggerated chemokine responses to intrahippocampal IL‐1β. Systemic LPS triggered microglial IL‐1β, astrocytic chemokines, IL‐6, and acute cognitive dysfunction, whereas IL‐1β disrupted hippocampal gamma rhythm, all selectively in APP/PS1 mice. Brains from AD patients with infection showed elevated IL‐1β and IL‐6 levels. Therefore, amyloid leaves the brain vulnerable to secondary inflammation at microglial, astrocytic, neuronal, and cognitive levels, and infection amplifies neuroinflammatory cytokine synthesis in humans. Exacerbation of neuroinflammation to produce deleterious outcomes like delirium and accelerated disease progression merits careful investigation in humans. [ABSTRACT FROM AUTHOR]

There have been significant advances in understanding human embryogenesis using human pluripotent stem cells (hPSCs) in conventional monolayer and 3D self-organized cultures. Thus, in vitro models have contributed to elucidate the molecular mechanisms for specification and differentiation during development. However, the molecular and functional spectrum of human pluripotency (i.e., intermediate states, pluripotency subtypes and regionalization) is still not fully understood. This review describes the mechanisms that establish and maintain pluripotency in human embryos and their differences with mouse embryos. Further, it describes a new pluripotent state representing a transition between naïve and primed pluripotency. This review also presents the data that divide pluripotency into substates expressing epiblast regionalization and amnion specification as well as primordial germ cells in primates. Finally, this work analyzes the amnion’s relevance as an “signaling center” for regionalization before the onset of gastrulation.

A major event in embryonic development is the rearrangement of epigenetic information as the somatic genome is reprogrammed for a new round of organismal development. Epigenetic data are held in chemical modifications on DNA and histones, and there are dramatic and dynamic changes in these marks during embryogenesis. However, the mechanisms behind this intricate process and how it is regulating and responding to embryonic development remain unclear. As embryos develop from totipotency to pluripotency, they pass through several distinct stages that can be captured permanently or transiently in vitro. Pluripotent naïve cells resemble the early epiblast, primed cells resemble the late epiblast, and blastomere-like cells have been isolated, although fully totipotent cells remain elusive. Experiments using these in vitro model systems have led to insights into chromatin changes in embryonic development, which has informed exploration of pre-implantation embryos. Intriguingly, human and mouse cells rely on different signaling and epigenetic pathways, and it remains a mystery why this variation exists. In this review, we will summarize the chromatin rearrangements in early embryonic development, drawing from genomic data from in vitro cell lines, and human and mouse embryos.

Pluripotent cells have been stabilized from pre- and post-implantation blastocysts, representing respectively naïve and primed stages of embryonic stem cells (ESCs) with distinct epigenetic, metabolic and transcriptomic features. Beside these two well characterized pluripotent stages, several intermediate states have been reported, as well as a small subpopulation of cells that have reacquired features of the 2C-embryo (2C-like cells) in naïve mouse ESC culture. Altogether, these represent a continuum of distinct pluripotency stages, characterized by metabolic transitions, for which we propose a new role for a long-known metabolite: succinate. Mostly seen as the metabolite of the TCA, succinate is also at the crossroad of several mitochondrial biochemical pathways. Its role also extends far beyond the mitochondrion, as it can be secreted, modify proteins by lysine succinylation and inhibit the activity of alpha-ketoglutarate-dependent dioxygenases, such as prolyl hydroxylase (PHDs) or histone and DNA demethylases. When released in the extracellular compartment, succinate can trigger several key transduction pathways after binding to SUCNR1, a G-Protein Coupled Receptor. In this review, we highlight the different intra- and extracellular roles that succinate might play in the fields of early pluripotency and embryo development.

RNA transcribed from enhancers, i.e., eRNA, has been suggested to directly activate transcription by recruiting transcription factors and co-activators. Although there have been specific examples of eRNA functioning in this way, it is not clear how general this may be. We find that the AT-hook of SWI/SNF preferentially binds RNA and, as part of the esBAF complex, associates with eRNA transcribed from intronic and intergenic regions. Our data suggest that SWI/SNF is globally recruited in cis by eRNA to cell-type-specific enhancers, representative of two distinct stages that mimic early mammalian development, and not at enhancers that are shared between the two stages. In this manner, SWI/SNF facilitates recruitment and/or activation of MLL3/4, p300/CBP, and Mediator to stage-specific enhancers and super-enhancers that regulate the transcription of metabolic and cell lineage priming-related genes. These findings highlight a connection between ATP-dependent chromatin remodeling and eRNA in cell identity and typical- and super-enhancer activation., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

Gonadal hormones may influence sexual activity by reducing anxiety. The basolateral amygdala (BLA) and prelimbic (PL) and infralimbic (IL) cortical regions comprise a loop that is related to fear, anxiety, and social behavior. In female ovariectomized rats, actions of estradiol, progesterone, and sequential estradiol and progesterone administration were explored in the open field test (OFT) and plus maze test (PMT) to evaluate signs of anxiety-like behavior. The three hormonal treatments reduced indicators of anxiety in the PMT but did not influence behavior in the OFT. In the same behaviorally tested rats under urethane anesthesia, single-unit extracellular recordings were obtained from the PL and IL during electrical stimulation of the BLA. The analysis of 250 ms peristimulus histograms showed that BLA stimulation produced two kinds of response. A small group of neurons increased their firing rate after BLA stimulation. Most neurons exhibited a reduction of spiking. Neurons that increased their firing rate after BLA stimulation did not show any difference with the hormonal treatments. In neurons that were inhibited by BLA stimulation, estradiol reduced the neuronal firing rate in the PL and IL, and progesterone alone and the sequential administration of estradiol followed by progesterone administration 24 h later (priming) increased the firing rate during the 240 ms before BLA stimulation. Analyses of responsivity of the PL and IL during electrical stimulation of the BLA indicated that estradiol, progesterone, and estradiol followed by progesterone administration 24 h later (priming) reduced inhibitory actions of the BLA on the PL but not IL. In the BLA-IL connection, progesterone exacerbated the inhibitory response. These findings indicate that anxiolytic actions of estradiol, progesterone, and estradiol followed by progesterone administration 24 h later (priming) correspond to lower BLA-PL responsivity. Actions of progesterone on BLA-IL responsivity appear to contribute to sexual activity by interacting with other forebrain structures that are also related to sexual receptivity. • Estradiol, progesterone, and sequential estradiol and progesterone administration produce anxiolytic actions. • Basolateral amygdala stimulation inhibits prelimbic and infralimbic region activity. • Estradiol, progesterone, and its sequential administration reduce amygdala inhibition of the prelimbic region. • Progesterone increases inhibitory actions of the basolateral amygdala on the infralimbic region. [ABSTRACT FROM AUTHOR]

The progression from naive through formative to primed in vitro pluripotent stem cell states recapitulates epiblast development in vivo during the peri-implantation period of mouse embryo development. Activation of the de novo DNA methyltransferases and reorganization of transcriptional and epigenetic landscapes are key events that occur during these pluripotent state transitions. However, the upstream regulators that coordinate these events are relatively underexplored. Here, using Zfp281 knockout mouse and degron knockin cell models, we identify the direct transcriptional activation of Dnmt3a/3b by ZFP281 in pluripotent stem cells. Chromatin co-occupancy of ZFP281 and DNA hydroxylase TET1, which is dependent on the formation of R-loops in ZFP281-targeted gene promoters, undergoes a "high-low-high" bimodal pattern regulating dynamic DNA methylation and gene expression during the naive-formative-primed transitions. ZFP281 also safeguards DNA methylation in maintaining primed pluripotency. Our study demonstrates a previously unappreciated role for ZFP281 in coordinating DNMT3A/3B and TET1 functions to promote pluripotent state transitions. [Display omitted] • ZFP281 activates Dnmt3a/3b in pluripotent stem cells in vitro and epiblast in vivo • ZFP281 and TET1 undergo bimodal chromatin occupancy in pluripotent state transitions • ZFP281 and TET1 chromatin binding depends on the formation of R-loops at promoters • ZFP281 is necessary for the establishment and maintenance of primed pluripotency The naive, formative, and primed pluripotent states and their interconversions recapitulate the pluripotency continuum during early development. Huang et al. investigate the transcriptional programs and identify an essential role for ZFP281 in coordinating DNMT3A/3B and TET1 to establish the DNA methylation and gene expression programs during the pluripotent state transitions. [ABSTRACT FROM AUTHOR]

Regenerative potential of mesenchymal stem/stromal cells (MSCs) has led to their application in various cellular therapies. Since in vivo these cells are present in very low numbers, they need expansion in culture to get clinically relevant numbers; however, such long-term ex vivo manipulation leads to loss of their regenerative capacity. Although use of naïve MSCs is still the most common approach used in various therapies, several strategies, both genetic and pharmacological, are being tried out to boost the regenerative capacity of in vitro expanded MSCs. Such manipulations are very commonly reported for regeneration of various tissues like bone, cartilage, kidney, pancreas, and others. Likewise, several efforts have been made to investigate priming of MSCs to enhance their immunoregulatory activity, but such efforts have not been made to the same extent for enhancing the efficacy of hematopoietic stem cell transplantation (HSCT). Development of such approaches for HSCT would not only be useful for enhancing the transplantation efficacy of cord blood cells, which are fewer in numbers, and aged HSCs, which could be functionally compromised, but also for genetically modified HSCs, which are likely to be both, fewer in number and functionally compromised. This review specifically deals with application of "primed" MSCs in the scenario of HSCT. [ABSTRACT FROM AUTHOR]

Microglia are the resident immune cells of the central nervous system that mediate the life and death of nervous tissue. During normal function, they exhibit a surveying phenotype and maintain vital functions in nervous tissue. In the event of injury or disease, chronic inflammation can result, wherein microglia develop a hyper-activated phenotype, shed their regenerative function, actively kill contiguous cells, and can partition injured tissue by initiating scar formation. With recoverable injury, microglia can develop a primed phenotype, where they appear to recover from an inflammatory event, but are limited in their support functions and show inappropriate responses to future injury often associated with neurodegenerative disorders. These microglial phenotypes were acutely recreated in vitro with potent pro- and anti-inflammatory treatments. Primary cultured microglia or mixed glia (microglia, astrocytes, and oligodendrocytes) were treated for 6 h with lipopolysaccharide (LPS). Recovery from an inflammatory state was modeled with 18-h treatment of the anti-inflammatory steroid dexamethasone. The cells were then treated for 24 h with interferon gamma (IFNγ) to detect inflammatory memory after recovery. Surveying was best represented in the untreated vehicle (Veh) cases and was characterized by negligible secretion of pro-inflammatory factors, limited expression of immune proteins such as induced nitric oxide synthase (iNOS), major histocompatibility complex class II (MHCII), relatively high expression of brain-derived and glial-derived neurotrophic factors (BDNF and GDNF), and thinly branched smaller microglia. Activation was noted in the LPS- and IFNγ-treated microglia with increased cytokines, NO, NGF, iNOS, proliferation, phagocytosis, reduced BDNF, and flattened round amoeboid-shaped microglia. Priming was observed to be an incomplete surveying restoration using dexamethasone from an activation comparison of LPS, IFNγ, and LPS/IFNγ. Dexamethasone treatments resulted in the most profound dysregulation of expression of NO, TNF, IL-1β, NGF, CD68, and MHCII as well as ramified morphology and uptake of myelin. These findings suggest microglial priming and hyper-activation may be effectively modeled in vitro to allow mechanistic investigations into these key cellular phenotypes. [ABSTRACT FROM AUTHOR]

CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against invading genetic elements, such as plasmids, bacteriophages and archaeal viruses. They consist of cas genes and CRISPR loci, which store genetic memories of previously encountered invaders as short sequences termed spacers. Spacers determine the specificity of CRISPR-Cas defence and immunity can be gained or updated by the addition of new spacers into CRISPR loci. There are two main routes to spacer acquisition, which are known as naïve and primed CRISPR adaptation. Naïve CRISPR adaptation involves the de novo formation of immunity, independent of pre-existing spacers. In contrast, primed CRISPR adaptation (priming) uses existing spacers to enhance the acquisition of new spacers. Priming typically results in spacer acquisition from locations near the site of target recognition by the existing (priming) spacer. Primed CRISPR adaptation has been observed in several type I CRISPR-Cas systems and it is potentially widespread. However, experimental evidence is unavailable for some subtypes, and for most systems, priming has only been shown in a small number of hosts. There is also no current evidence of priming by other CRISPR-Cas types. Here, we used a bioinformatic approach to search for evidence of priming in diverse CRISPR-Cas systems. By analysing the clustering of spacers acquired from phages, prophages and archaeal viruses, including strand and directional biases between subsequently acquired spacers, we demonstrate that two patterns of primed CRISPR adaptation dominate in type I systems. In addition, we find evidence of a priming-like pathway in type II CRISPR-Cas systems. [ABSTRACT FROM AUTHOR]

During early mammalian development, transient pools of pluripotent cells emerge that can be immortalised upon stem cell derivation. The pluripotent state, ‘naïve’ or ‘primed’, depends on the embryonic stage and derivation conditions used. Here we analyse the temporal gene expression patterns of mouse, cattle and porcine embryos at stages that harbour different types of pluripotent cells. We document conserved and divergent traits in gene expression, and identify predictor genes shared across the species that are associated with pluripotent states in vivo and in vitro. Amongst these are the pluripotency-linked genes Klf4 and Lin28b. The novel genes discovered include naïve- (Spic, Scpep1 and Gjb5) and primed-associated (Sema6a and Jakmip2) genes as well as naïve to primed transition genes (Dusp6 and Trip6). Both Gjb5 and Dusp6 play a role in pluripotency since their knockdown results in differentiation and downregulation of key pluripotency genes. Our interspecies comparison revealed new insights of pluripotency, pluripotent stem cell identity and a new molecular criterion for distinguishing between pluripotent states in various species, including human.

Pluripotent stem cells (PSCs) lie at the heart of modern regenerative medicine due to their properties of unlimited self-renewal in vitro and their ability to differentiate into cell types representative of the three embryonic germ layers--mesoderm, ectoderm and endoderm. The derivation of induced PSCs bypasses ethical concerns associated with the use of human embryonic stem cells and also enables personalized cell-based therapies. To exploit their regenerative potential, it is essential to have a firm understanding of the molecular processes associated with their induction from somatic cells. This understanding serves two purposes: first, to enable efficient, reliable and cost-effective production of excellent quality induced PSCs and, second, to enable the derivation of safe, goodmanufacturing practice-grade transplantable donor cells. Here, we review the reprogramming process of somatic cells into induced PSCs and associated mechanisms with emphasis on self-renewal, epigenetic control, mitochondrial bioenergetics, sub-states of pluripotency, naive ground state, naive and primed. A meta-analysis identified genes expressed exclusively in the inner cell mass and in the naive but not in the primed pluripotent state.We propose these as additional biomarkers defining naive PSCs. This article is part of the theme issue 'Designer human tissue: coming to a lab near you'. [ABSTRACT FROM AUTHOR]

The RNA-binding protein Musashi-1 (MSI1) regulates the proliferation and differentiation of adult stem cells. However, its role in embryonic stem cells (ESCs) and early embryonic development remains poorly understood. Here, we report the presence of short C-terminal MSI1 (MSI1-C) proteins in early mouse embryos and mouse ESCs, but not in human ESCs, under conventional culture conditions. In mouse embryos and mESCs, deletion of MSI1-C together with full-length MSI1 causes early embryonic developmental arrest and pluripotency dissolution. MSI1-C is induced upon naive induction and facilitates hESC naive pluripotency acquisition, elevating the pluripotency of primed hESCs toward a formative-like state. MSI1-C proteins are nuclear localized and bind to RNAs involved in DNA-damage repair (including MLH1 , BRCA1 , and MSH2), conferring on hESCs better survival in human-mouse interspecies cell competition and prolonged ability to form blastoids. This study identifies MSI1-C as an essential regulator in ESC pluripotency states and early embryonic development. [Display omitted] • MSI1 is localized in the nucleus of naive ESCs but in the cytoplasm of primed ESCs • Short MSI1-C in naive pluripotency state regulates mRNA stability of pluripotency genes • MSI1-C facilitates primed-to-naive state transition of primed hESCs • MSI1-C binds to RNAs and regulates DNA-damage repair and stress resistance Chen et al. report the presence of short Musashi-1 protein (MSI1-C) that regulates the naive pluripotency of embryonic stem cells and early development. MSI1-C promotes the acquisition of naive pluripotency in human embryonic cells by regulating mRNA stability and DNA-damage repair and increasing stress resistance. [ABSTRACT FROM AUTHOR]

Pluripotency defines the propensity of a cell to differentiate into, and generate, all somatic, as well as germ cells. The epiblast of the early mammalian embryo is the founder population of all germ layer derivatives and thus represents the bona fide in vivo pluripotent cell population. The so-called pluripotent state spans several days of development and is lost during gastrulation as epiblast cells make fate decisions towards a mesoderm, endoderm or ectoderm identity. It is now widely recognized that the features of the pluripotent population evolve as development proceeds from the pre- to post-implantation period, marked by distinct transcriptional and epigenetic signatures. During this period of time epiblast cells mature through a continuum of pluripotent states with unique properties. Aspects of this pluripotent continuum can be captured in vitro in the form of stable pluripotent stem cell types. In this review we discuss the continuum of pluripotency existing within the mammalian embryo, using the mouse as a model, and the cognate stem cell types that can be derived and propagated in vitro. Furthermore, we speculate on embryonic stage-specific characteristics that could be utilized to identify novel, developmentally relevant, pluripotent states. [ABSTRACT FROM AUTHOR]

The naïve state of pluripotency is actively being explored by a number of labs. There is some controversy in the field as to the true identity of naïve human pluripotent cells as they are not exact mirrors of the mouse. The various reports published, although in basic agreement, present discrepancies in the characterization of the various lines, which likely reflect the etiology of these lines. The primary lesson learned from these contributions is that a human naïve state reflecting the preimplantation human is likely to exist. The essential factors that will universally maintain the naïve state in human cells in vitro are not yet fully understood. These first need to be identified in order to describe the definitive characteristics of this state. Comparisons of naïve and primed human pluripotent cells have also highlighted consistencies between states and broadened our understanding of embryonic metabolism, epigenetic change required for development, embryonic DNA repair strategies and embryonic expression dynamics. S tem C ells 2017;35:35-41 [ABSTRACT FROM AUTHOR]

The progression from naive through formative to primed in vitro pluripotent stem cell states recapitulates the development of the epiblast in vivo during the peri-implantation period of mammalian development. Activation of the de novo DNA methyltransferases and reorganization of transcriptional and epigenetic landscapes are key events occurring during these pluripotent state transitions. However, the upstream regulators that coordinate these events are relatively underexplored. Here, using Zfp281 knockout mouse and degron knock-in cell models, we uncover the direct transcriptional activation of Dnmt3a/3b by ZFP281 in pluripotent stem cells. Chromatin co-occupancy of ZFP281 and DNA hydroxylase TET1, dependent on the formation of R loops in ZFP281-targeted gene promoters, undergoes a "high-low-high" bimodal pattern regulating dynamic DNA methylation and gene expression during the naïive-formative-primed transitions. ZFP281 also safeguards DNA methylation in maintaining primed pluripotency. Our study demonstrates a previously unappreciated role for ZFP281 in coordinating DNMT3A/3B and TET1 functions to promote pluripotent state transitions.

Prior to differentiation, embryonic stem (ES) cells in culture are maintained in a so-called 'undifferentiated' state, allowing derivation of multiple downstream cell lineages when induced in a directed manner, which in turn grants these cells their 'pluripotent' state. The current work is based on a simple observation that the initial culture condition for maintaining mouse ES cells in an 'undifferentiated' state does impact on the differentiation propensity of these cells, in this case to a neuronal fate. We point out the importance in judging the 'pluripotency' of a given stem cell culture, as this clearly demonstrated that the 'undifferentiated' state of these cells is not necessarily a 'pluripotent' state, even for a widely used mouse ES cell line. We partly attribute this difference in the initial value of ES cells to the naïve-to-primed status of pluripotency, which in turn may affect early events of the differentiation in vitro. [ABSTRACT FROM AUTHOR]

Universally primed (UP) PCR analysis was used to characterize strains of Trichoderma spp., which constitute the active ingredients of commercial products for biocontrol of phytopathogens. Several UP primers were able to generate distinct and reproducible fingerprints for each strain, allowing them to be differentiated from a collection of other Trichoderma spp. strains. In order to test whether the UP-PCR method in combination with dilution plating could be useful to detect and enumerate propagules of biocontrol strains of Trichoderma spp. when applied on a commercial scale, sampling was carried out in three commercial glasshouse operations. Dilution plating was carried out using a semi-selective medium and a number of the Trichoderma spp. recovered were subjected to UP-PCR analysis with one selected UP primer. The results showed that the method could be used to identify the isolates from the biocontrol products applied in the different glasshouses. Furthermore, it was found that a biocontrol strain also colonized an untreated bench in one of the glasshouses. The combined methods enable verification of the active ingredient concentration, facilitating monitoring establishment and spread of the Trichoderma strains. [ABSTRACT FROM AUTHOR]

Cell-surface proteins provide excellent biomarkers to identify specific cell types and resolve heterogeneous cell populations. The analysis of cell-surface proteins by flow cytometry produces robust and quantitative information with single-cell resolution, and allows live target cells to be purified and characterized or re-cultured. Studies using antibody screens, proteomics, and candidate analysis have identified a comprehensive set of proteins that are expressed on the surface of naïve and primed human pluripotent stem cells. These findings have led to the development of suitable protein markers and antibodies to accurately distinguish between these two cell types. Here, a detailed protocol is provided that uses multi-color flow cytometry to analyze cell-surface protein expression in naïve and primed human pluripotent stem cells. This method enables the unambiguous identification of pluripotent cell types and the opportunity to sort target cells including during cell state transitions. The protocol can be combined to additionally investigate the expression of reporter genes and other informative features, such as DNA content., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Neuroinflammation contributes to Alzheimer's disease (AD) progression. Secondary inflammatory insults trigger delirium and can accelerate cognitive decline. Individual cellular contributors to this vulnerability require elucidation. Using APP/PS1 mice and AD brain, we studied secondary inflammatory insults to investigate hypersensitive responses in microglia, astrocytes, neurons, and human brain tissue. The NLRP3 inflammasome was assembled surrounding amyloid beta, and microglia were primed, facilitating exaggerated interleukin-1β (IL-1β) responses to subsequent LPS stimulation. Astrocytes were primed to produce exaggerated chemokine responses to intrahippocampal IL-1β. Systemic LPS triggered microglial IL-1β, astrocytic chemokines, IL-6, and acute cognitive dysfunction, whereas IL-1β disrupted hippocampal gamma rhythm, all selectively in APP/PS1 mice. Brains from AD patients with infection showed elevated IL-1β and IL-6 levels. Therefore, amyloid leaves the brain vulnerable to secondary inflammation at microglial, astrocytic, neuronal, and cognitive levels, and infection amplifies neuroinflammatory cytokine synthesis in humans. Exacerbation of neuroinflammation to produce deleterious outcomes like delirium and accelerated disease progression merits careful investigation in humans., (© 2021 The Authors. Alzheimer's & Dementia published by Wiley Periodicals LLC on behalf of Alzheimer's Association.)