Pursuing totipotency: authentic totipotent stem cells in culture.
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ABSTRACT:
SUBMITTER: Malik V
PROVIDER: S-EPMC9197991 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Embryonic stem cells (ESCs) are derived from mammalian embryos during the transition from totipotency, when individual blastomeres can make all lineages, to pluripotency, when they are competent to make only embryonic lineages. ESCs maintained with inhibitors of MEK and GSK3 (2i) are thought to represent an embryonically restricted ground state. However, we observed heterogeneous expression of the extraembryonic endoderm marker Hex in 2i-cultured embryos, suggesting that 2i blocked development prior to epiblast commitment. Similarly, 2i ESC cultures were heterogeneous and contained a Hex-positive fraction primed to differentiate into trophoblast and extraembryonic endoderm. Single Hex-positive ESCs coexpressed epiblast and extraembryonic genes and contributed to all lineages in chimeras. The cytokine LIF, necessary for ESC self-renewal, supported the expansion of this population but did not directly support Nanog-positive epiblast-like ESCs. Thus, 2i and LIF support a totipotent state comparable to early embryonic cells that coexpress embryonic and extraembryonic determinants.
| S-EPMC3701323 | biostudies-literature
Project description:Pig embryonic stem cells (ESCs) have been considered as an important candidate for preclinical researches on human therapy. However, the lack of understanding of pig pluripotent networks has hampered establishment of authentic pig ESCs. Here, we report that FGF2, ACTVIN, and WNT signaling is essential for maintaining pig pluripotency in vitro. Pig ESC lines derived by stimulating three signlaings formed colonies of flattened monolayer morphology. Newly derived ESCs were stably maintained over an extended period, and were capable of forming teratomas comprising three germ layers. Immunostaining showed that the stem cells expressed pluripotency markers including OCT4, SOX2, NANOG, SSEA1, and SSEA4. Transcriptome analysis showed that pig ESCs were developmentally similar to late epiblasts of preimplantation embryos and in terms of biological functions resembled human rather than mouse ESCs. However, the pig ESCs had distinct features such as coexpression of SSEA1 and SSEA4, two active X chromosomes, and a unique transcriptional pattern. In conclusion, we successfully derived authentic pig ESCs using novel cell culture conditions. Our findings will facilitate both the development of large animal models for human stem cell therapy and the generation of pluripotent stem cells from other domestic animals for agricultural use.
2019-07-08 | GSE120031 | GEO
Project description:The cell-fate transition between pluripotent and totipotent states determines embryonic development and the first cell-lineage segregation. However, limited by the scarcity of totipotent embryos, regulators on this transition remain largely elusive. A novel model to study the transition has been recently established, named the 2-cell-like (2C-like) model. The 2C-like cells are rare totipotent-like cells in the mouse embryonic stem cell (mESC) culture. Pluripotent mESCs can spontaneously transit into and out of the 2C-like state. We previously dissected the transcriptional roadmap of the transition. In this study, we revealed that Zfp281 is a novel regulator for the pluripotent-to-totipotent transition in mESCs. Zfp281 is a transcriptional factor involved in the cell-fate transition. Our study shows that Zfp281 represses transcripts upregulated during the 2C-like transition via Tet1 and consequentially inhibits mESCs from transiting into the 2C-like state. Interestingly, we found that the inhibitory effect of Zfp281 on the 2C-like transition leads to an impaired 2C-like-transition ability in primed-state mESCs. Altogether, our study reveals a novel mediator for the pluripotent-to-totipotent state transition in mESCs and provides insights into the dynamic transcriptional control of the transition.
| S-EPMC9163740 | biostudies-literature
Project description:Totipotency refers to the ability of a cell to generate all of the cell types of an organism. Unlike pluripotency, the establishment of totipotency is poorly understood. In mouse embryonic stem cells, Dux drives a small percentage of cells into a totipotent state by expressing 2-cell-embryo-specific transcripts. To understand how this transition takes place, we performed single-cell RNA-seq, which revealed a two-step transcriptional reprogramming process characterized by downregulation of pluripotent genes in the first step and upregulation of the 2-cell-embryo-specific elements in the second step. To identify factors controlling the transition, we performed a CRISPR-Cas9-mediated screen, which revealed Myc and Dnmt1 as two factors preventing the transition. Mechanistic studies demonstrate that Myc prevents downregulation of pluripotent genes in the first step, while Dnmt1 impedes 2-cell-embryo-specific gene activation in the second step. Collectively, the findings of our study reveal insights into the establishment and regulation of the totipotent state in mouse embryonic stem cells.
| S-EPMC7137718 | biostudies-literature
Project description:Cellular totipotency is critical for whole-organism generation, yet how totipotency is established remains poorly illustrated. Abundant transposable elements (TEs) are activated in totipotent cells, which is critical for embryonic totipotency. Here, we show that the histone chaperone RBBP4, but not its homolog RBBP7, is indispensable for maintaining the identity of mouse embryonic stem cells (mESCs). Auxin-induced degradation of RBBP4, but not RBBP7, reprograms mESCs to the totipotent 2C-like cells. Also, loss of RBBP4 enhances transition from mESCs to trophoblast cells. Mechanistically, RBBP4 binds to the endogenous retroviruses (ERVs) and functions as an upstream regulator by recruiting G9a to deposit H3K9me2 on ERVL elements, and recruiting KAP1 to deposit H3K9me3 on ERV1/ERVK elements, respectively. Moreover, RBBP4 facilitates the maintenance of nucleosome occupancy at the ERVK and ERVL sites within heterochromatin regions through the chromatin remodeler CHD4. RBBP4 depletion leads to the loss of the heterochromatin marks and activation of TEs and 2C genes. Together, our findings illustrate that RBBP4 is required for heterochromatin assembly and is a critical barrier for inducing cell fate transition from pluripotency to totipotency.
| S-EPMC10287929 | biostudies-literature
Project description:The 3D genome organization is crucial for gene regulation. Although recent studies have revealed a uniquely relaxed genome conformation in totipotent early blastomeres of both fertilized and cloned embryos, how weakened higher-order chromatin structure is functionally linked to totipotency acquisition remains elusive. Using low-input Hi-C, ATAC-seq and ChIP-seq, we systematically examined the dynamics of 3D genome and epigenome during pluripotent to totipotent-like state transition in mouse embryonic stem cells (ESCs). The spontaneously converted 2-cell-embryo-like cells (2CLCs) exhibited more relaxed chromatin architecture compared to ESCs, including global weakening of both enhancer-promoter interactions and TAD insulation. While the former correlated with inactivation of ESC enhancers and down-regulation of pluripotent genes, the latter might facilitate contacts between the putative new enhancers arising in 2CLCs and neighboring 2C genes. Importantly, disruption of chromatin organization by depleting CTCF or the cohesin complex promoted the ESC to 2CLC transition. Our results thus establish a critical role of 3D genome organization in totipotency acquisition.
| S-EPMC8643704 | biostudies-literature
Project description:Background and aimsUnderstanding the fate and dynamics of cells during callus formation is essential to understanding totipotency and the mechanisms of somatic embryogenesis. Here, the fate of leaf explant cells during the development of embryogenic callus was investigated in the model legume Medicago truncatula.MethodsCallus development was examined from cultured leaf explants of the highly regenerable genotype Jemalong 2HA (2HA) and from mesophyll protoplasts of 2HA and wild-type Jemalong. Callus development was studied by histology, manipulation of the culture system, detection of early production of reactive oxygen species and visualization of SERK1 (SOMATIC EMBRYO RECEPTOR KINASE1) gene expression.Key resultsCallus formation in leaf explants initiates at the cut surface and within veins of the explant. The ontogeny of callus development is dominated by the division and differentiation of cells derived from pluripotent procambial cells and from dedifferentiated mesophyll cells. Procambium-derived cells differentiated into vascular tissue and rarely formed somatic embryos, whereas dedifferentiated mesophyll cells were competent to form somatic embryos. Interestingly, explants incubated adaxial-side down had substantially less cell proliferation associated with veins yet produced similar numbers of somatic embryos to explants incubated abaxial-side down. Somatic embryos mostly formed on the explant surface originally in contact with the medium, while in protoplast microcalli, somatic embryos only fully developed once at the surface of the callus. Mesophyll protoplasts of 2HA formed embryogenic callus while Jemalong mesophyll protoplasts produced callus rich in vasculature.ConclusionsThe ontogeny of embryogenic callus in M. truncatula relates to explant orientation and is driven by the dynamics of pluripotent procambial cells, which proliferate and differentiate into vasculature. The ontogeny is also related to de-differentiated mesophyll cells that acquire totipotency and form the majority of embryos. This contrasts with other species where totipotent embryo-forming initials mostly originate from procambial cells.
| S-EPMC3064535 | biostudies-literature
Project description:Derivation of authentic porcine embryonic stem cells using defined culture conditions
| PRJNA491422 | ENA
Project description:Stem cells occupy variable environments where they must distinguish stochastic fluctuations from developmental cues. Here, we use optogenetics to investigate how the pluripotency network in embryonic stem (ES) cells achieves a robust response to differentiation cues but not to gene expression fluctuations. We engineered ES cells in which we could quantitatively ontrol the endogenous mechanism of neural differentiation through a light-inducible Brn2 transgene and monitor differentiation status through a genome-integrated Nanog-GFP reporter. By exposing cells to pulses of Brn2, we find that the pluripotency network rejects Brn2 inputs that are below specific magnitude or duration thresholds, but allows rapid differentiation when both thresholds are satisfied. The filtering properties of the network arise through its positive feedback architecture and the intrinsic half-life of Nanog, which determines the duration threshold in the network. Together our results suggest that the dynamic properties of positive-feedback networks might determine how inputs are classified as signal or noise by stem cells.
| S-EPMC4576702 | biostudies-literature
Project description:Our perception of the role of the previously considered 'selfish' or 'junk' DNA has been dramatically altered in the past 20 years or so. A large proportion of this non-coding part of mammalian genomes is repetitive in nature, classified as either satellites or transposons. While repetitive elements can be termed selfish in terms of their amplification, such events have surely been co-opted by the host, suggesting by itself a likely altruistic function for the organism at the subject of such natural selection. Indeed numerous examples of transposons regulating the functional output of the host genome have been documented. Transposons provide a powerful framework for large-scale relatively rapid concerted regulatory activities with the ability to drive evolution. Mammalian totipotency has emerged as one key stage of development in which transposon-mediated regulation of gene expression has taken centre stage in the past few years. During this period, large-scale (epigenetic) reprogramming must be accomplished in order to activate the host genome. In mice and men, one particular element murine endogenous retrovirus with leucine tRNA primer (MERVL) (and its counterpart human ERVL (HERVL)) appears to have acquired roles as a key driving force in this process. Here, I will discuss and interpret the current knowledge and its implications regarding the role of transposons, particularly of long interspersed nuclear elements (LINE-1s) and endogenous retroviruses (ERVs), in the regulation of totipotency. This article is part of a discussion meeting issue 'Crossroads between transposons and gene regulation'.
| S-EPMC7061993 | biostudies-literature