In the mammalian embryo, epiblast cells must exit the naïve state and acquire formative pluripotency. This cell state transition is recapitulated by mouse embryonic stem cells (ESCs), which undergo ...pluripotency progression in defined conditions in vitro. However, our understanding of the molecular cascades and gene networks involved in the exit from naïve pluripotency remains fragmentary. Here, we employed a combination of genetic screens in haploid ESCs, CRISPR/Cas9 gene disruption, large‐scale transcriptomics and computational systems biology to delineate the regulatory circuits governing naïve state exit. Transcriptome profiles for 73 ESC lines deficient for regulators of the exit from naïve pluripotency predominantly manifest delays on the trajectory from naïve to formative epiblast. We find that gene networks operative in ESCs are also active during transition from pre‐ to post‐implantation epiblast in utero. We identified 496 naïve state‐associated genes tightly connected to the in vivo epiblast state transition and largely conserved in primate embryos. Integrated analysis of mutant transcriptomes revealed funnelling of multiple gene activities into discrete regulatory modules. Finally, we delineate how intersections with signalling pathways direct this pivotal mammalian cell state transition.
SYNOPSIS
Combining a haploid transposon‐based screen and systems biology, this resource work identifies gene networks controlling the exit of mouse embryonic stem cells (ESC) from naïve pluripotency, and delineates signalling intersections involved.
Transcriptional characterization of 73 differentiation‐defective ESC lines defines discrete gene modules and signalling inputs essential for the exit from naïve pluripotency.
Genetic depletion of specific exit genes in ESCs results in increased molecular similarity to the pre‐implantation epiblast in utero.
Identification of 496 naïve state‐associated genes establishes an extended naïve pluripotency network conserved in primate embryos.
The transcription factor Klf2 is only weakly wired into the mouse naïve pluripotency network.
An extensive mutagenesis screen identifies regulatory gene modules governing exit from naïve mammalian pluripotency.
Recently naive human pluripotent stem cells (hPSCs) have been described that relate to an earlier stage of development than conventional hPSCs. Naive hPSCs remain challenging to generate and ...authenticate, however. Here we report that Sushi Containing Domain 2 (SUSD2) is a robust cell-surface marker of naive hPSCs in the embryo and in vitro. SUSD2 transcripts are enriched in the pre-implantation epiblast of human blastocysts and immunostaining shows localization of SUSD2 to KLF17-positive epiblast cells. SUSD2 mRNA is strongly expressed in naive hPSCs but is negligible in other hPSCs. SUSD2 immunostaining of live or fixed cells provides unambiguous discrimination of naive versus conventional hPSCs. SUSD2 staining or flow cytometry enable monitoring of naive hPSCs in maintenance culture, and their isolation and quantification during resetting of conventional hPSCs or somatic cell reprogramming. Thus SUSD2 is a powerful non-invasive tool for reliable identification and purification of the naive hPSC phenotype.
•SUSD2 is a specific cell-surface marker of naive epiblast in the human blastocyst•SUSD2 staining unambiguously discriminates naive from other hPSCs•SUSD2 facilitates isolation of naive hPSCs during resetting or reprogramming
In this article, Guo and colleagues identified SUSD2 as a cell-surface marker highly enriched in naive epiblast in the human blastocyst. SUSD2 staining unambiguously discriminates naive from other hPSCs and provides a powerful tool for identification and isolation of human naive stem cells during resetting of conventional hPSCs or somatic cell reprogramming.
Lineage segregation in the mouse embryo is a finely controlled process dependent upon coordination of signalling pathways and transcriptional responses. Here we employ a conditional deletion system ...to investigate embryonic patterning and lineage specification in response to loss of Oct4. We first observe ectopic expression of Nanog in Oct4-negative postimplantation epiblast cells. The expression domains of lineage markers are subsequently disrupted. Definitive endoderm expands at the expense of mesoderm; the anterior-posterior axis is positioned more distally and an ectopic posterior-like domain appears anteriorly, suggesting a role for Oct4 in maintaining the embryonic axis. Although primitive streak forms in the presumptive proximal-posterior region, epithelial-to-mesenchymal transition is impeded by an increase of E-cadherin, leading to complete tissue disorganisation and failure to generate germ layers. In explant and
differentiation assays, Oct4 mutants also show upregulation of E-cadherin and Foxa2, suggesting a cell-autonomous phenotype. We confirm requirement for Oct4 in self-renewal of postimplantation epiblast
Our results indicate a role for Oct4 in orchestrating multiple fates and enabling expansion, correct patterning and lineage choice in the postimplantation epiblast.
Cardiac hypertrophy, initially an adaptive response of the myocardium to stress, can progress to heart failure. The epigenetic signature underlying this phenomenon is poorly understood. Here, we ...report on the genome-wide distribution of seven histone modifications in adult mouse cardiomyocytes subjected to a prohypertrophy stimulus in vivo. We found a set of promoters with an epigenetic pattern that distinguishes specific functional classes of genes regulated in hypertrophy and identified 9,207 candidate active enhancers whose activity was modulated. We also analyzed the transcriptional network within which these genetic elements act to orchestrate hypertrophy gene expression, finding a role for myocyte enhancer factor (MEF)2C and MEF2A in regulating enhancers. We propose that the epigenetic landscape is a key determinant of gene expression reprogramming in cardiac hypertrophy and provide a basis for understanding the role of chromatin in regulating this phenomenon.
The sympathetic nervous system plays a fundamental role in the regulation of myocardial function. During chronic pressure overload, overactivation of the sympathetic nervous system induces the ...release of catecholamines, which activate β-adrenergic receptors in cardiomyocytes and lead to increased heart rate and cardiac contractility. However, chronic stimulation of β-adrenergic receptors leads to impaired cardiac function, and β-blockers are widely used as therapeutic agents for the treatment of cardiac disease. MicroRNA-133 (miR-133) is highly expressed in the myocardium and is involved in controlling cardiac function through regulation of messenger RNA translation/stability.
To determine whether miR-133 affects β-adrenergic receptor signaling during progression to heart failure.
Based on bioinformatic analysis, β1-adrenergic receptor (β1AR) and other components of the β1AR signal transduction cascade, including adenylate cyclase VI and the catalytic subunit of the cAMP-dependent protein kinase A, were predicted as direct targets of miR-133 and subsequently validated by experimental studies. Consistently, cAMP accumulation and activation of downstream targets were repressed by miR-133 overexpression in both neonatal and adult cardiomyocytes following selective β1AR stimulation. Furthermore, gain-of-function and loss-of-function studies of miR-133 revealed its role in counteracting the deleterious apoptotic effects caused by chronic β1AR stimulation. This was confirmed in vivo using a novel cardiac-specific TetON-miR-133 inducible transgenic mouse model. When subjected to transaortic constriction, TetON-miR-133 inducible transgenic mice maintained cardiac performance and showed attenuated apoptosis and reduced fibrosis compared with control mice.
miR-133 controls multiple components of the β1AR transduction cascade and is cardioprotective during heart failure.
In contrast to conventional human pluripotent stem cells (hPSCs) that are related to post-implantation embryo stages, naive hPSCs exhibit features of pre-implantation epiblast. Naive hPSCs are ...established by resetting conventional hPSCs, or are derived from dissociated embryo inner cell masses. Here we investigate conditions for transgene-free reprogramming of human somatic cells to naive pluripotency. We find that Wnt inhibition promotes RNA-mediated induction of naive pluripotency. We demonstrate application to independent human fibroblast cultures and endothelial progenitor cells. We show that induced naive hPSCs can be clonally expanded with a diploid karyotype and undergo somatic lineage differentiation following formative transition. Induced naive hPSC lines exhibit distinctive surface marker, transcriptome, and methylome properties of naive epiblast identity. This system for efficient, facile, and reliable induction of transgene-free naive hPSCs offers a robust platform, both for delineation of human reprogramming trajectories and for evaluating the attributes of isogenic naive versus conventional hPSCs.
•Generation of transgene-free human naive iPSCs by RNA-mediated reprogramming•Wnt inhibition facilitates naive iPSC production and expansion•Naive iPSCs retain a diploid karyotype•Naive iPSCs are transcriptomically related to pre-implantation human epiblast
Molecular reprogramming can induce different states of pluripotency. In this paper, Ge Guo and colleagues report that naive stem cells related to the blastocyst stage human embryo can be generated reliably from human somatic cells using RNA delivery of reprogramming factors. Reprogramming is enhanced by inhibition of the Wnt pathway, which also stabilizes the human naive state.
Previous analysis of RNA sequencing (RNA-seq) data from human naive pluripotent stem cells reported multiple point “mutations” in cancer-related genes and implicated selective culture conditions. We ...observed, however, that those mutations were only present in co-cultures with mouse feeder cells. Inspection of reads containing the polymorphisms revealed complete identity to the mouse reference genome. After we filtered reads to remove sequences of mouse origin, the actual incidence of oncogenic polymorphisms arising in naive pluripotent stem cells is close to zero.
•Culture conditions for naive stem cells do not select for cancer-related mutations•Contaminating sequences in RNA-seq data can be misidentified as point mutations•Filtering to remove mouse sequences eliminates most mutations•Remaining mutations in naive stem cells are carried over from primed stem cells
Acquisition of pathogenic mutations during in vitro propagation is a concern for applications of pluripotent stem cells. In this paper, Stirparo and colleagues show that detection of multiple mutations in naïve stem cells by RNA-seq analysis is due to contamination with mouse sequences. The actual cancer mutation incidence is low.
Pluripotent cells emerge as a naive founder population in the blastocyst, acquire capacity for germline and soma formation, and then undergo lineage priming. Mouse embryonic stem cells (ESCs) and ...epiblast-derived stem cells (EpiSCs) represent the initial naive and final primed phases of pluripotency, respectively. Here, we investigate the intermediate formative stage. Using minimal exposure to specification cues, we derive stem cells from formative mouse epiblast. Unlike ESCs or EpiSCs, formative stem (FS) cells respond directly to germ cell induction. They colonize somatic tissues and germline in chimeras. Whole-transcriptome analyses show similarity to pre-gastrulation formative epiblast. Signal responsiveness and chromatin accessibility features reflect lineage capacitation. Furthermore, FS cells show distinct transcription factor dependencies, relying critically on Otx2. Finally, FS cell culture conditions applied to human naive cells or embryos support expansion of similar stem cells, consistent with a conserved staging post on the trajectory of mammalian pluripotency.
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•Derivation of mouse formative pluripotent stem cells from pre-gastrulation epiblast•Formative stem cells are competent for germline induction•Formative stem cells can contribute to chimeras•Candidate human FS cells derived using similar culture conditions to mouse
Three stages of pluripotency have been proposed: naive, formative, and primed. Kinoshita and colleagues derived stem cells with properties anticipated for formative pluripotency by culturing mouse epiblast under conditions of low growth factor stimulation. Application to human embryos resulted in propagation of similar stem cells.
Classic embryological experiments have established that the early mouse embryo develops via sequential lineage bifurcations. The first segregated lineage is the trophectoderm, essential for ...blastocyst formation. Mouse naive epiblast and derivative embryonic stem cells are restricted accordingly from producing trophectoderm. Here we show, in contrast, that human naive embryonic stem cells readily make blastocyst trophectoderm and descendant trophoblast cell types. Trophectoderm was induced rapidly and efficiently by inhibition of ERK/mitogen-activated protein kinase (MAPK) and Nodal signaling. Transcriptome comparison with the human embryo substantiated direct formation of trophectoderm with subsequent differentiation into syncytiotrophoblast, cytotrophoblast, and downstream trophoblast stem cells. During pluripotency progression lineage potential switches from trophectoderm to amnion. Live-cell tracking revealed that epiblast cells in the human blastocyst are also able to produce trophectoderm. Thus, the paradigm of developmental specification coupled to lineage restriction does not apply to humans. Instead, epiblast plasticity and the potential for blastocyst regeneration are retained until implantation.
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•Human naive pluripotent stem cells form blastocyst trophectoderm directly•Unlike in mouse, human naive epiblast regenerates trophectoderm•Inhibition of ERK and Nodal drives trophectoderm differentiation•Potency changes from trophectoderm to amnion during pluripotency progression
Human pluripotent stem cells (hPSCs) exist in naive or primed states, but it has been unclear whether these correspond to distinct developmental potencies. Here, Guo et al. show that naive hPSCs differentiate into trophectoderm, the founder tissue of the placenta, whereas primed hPSCs have lost this potential and instead form the amnion.
The epidemic of heart failure has stimulated interest in understanding cardiac regeneration. Evidence has been reported supporting regeneration via transplantation of multiple cell types, as well as ...replication of postmitotic cardiomyocytes. In addition, the adult myocardium harbors endogenous c-kit(pos) cardiac stem cells (eCSCs), whose relevance for regeneration is controversial. Here, using different rodent models of diffuse myocardial damage causing acute heart failure, we show that eCSCs restore cardiac function by regenerating lost cardiomyocytes. Ablation of the eCSC abolishes regeneration and functional recovery. The regenerative process is completely restored by replacing the ablated eCSCs with the progeny of one eCSC. eCSCs recovered from the host and recloned retain their regenerative potential in vivo and in vitro. After regeneration, selective suicide of these exogenous CSCs and their progeny abolishes regeneration, severely impairing ventricular performance. These data show that c-kit(pos) eCSCs are necessary and sufficient for the regeneration and repair of myocardial damage.
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