Network analysis of transcriptomic data uncovers molecular signatures and the interplay of mRNAs, lncRNAs, and miRNAs in human embryonic stem cells.

Growing evidence has shown that besides the protein coding genes, the non-coding elements of the genome are indispensable for maintaining the property of self-renewal in human embryonic stem cells and in cell fate determination. However, the regulatory mechanisms and the landscape of interactions between the coding and non-coding elements is poorly understood. In this work, we used weighted gene co-expression network analysis (WGCNA) on transcriptomic data retrieved from RNA-seq and small RNA-seq experiments and reconstructed the core human pluripotency network (called PluriMLMiNet) consisting of 375 mRNA, 57 lncRNA and 207 miRNAs. Furthermore, we derived networks specific to the naïve and primed states of human pluripotency (called NaiveMLMiNet and PrimedMLMiNet respectively) that revealed a set of molecular markers (RPS6KA1, ZYG11A, ZNF695, ZNF273, and NLRP2 for naive state, and RAB34, TMEM178B, PTPRZ1, USP44, KIF1A and LRRN1 for primed state) which can be used to distinguish the pluripotent state from the non-pluripotent state and also to identify the intra-pluripotency states (i.e., naïve and primed state). The lncRNA DANT1 was found to be a crucial as it formed a bridge between the naive and primed state-specific networks. Analysis of the genes neighbouring DANT1 suggested its possible role as a competing endogenous RNA (ceRNA) for the induction and maintenance of human pluripotency. This was computationally validated by predicting the missing DANT1-miRNA interactions to complete the ceRNA circuit. Here we first report that DANT1 might harbour binding sites for miRNAs hsa-miR-30c-2-3p, hsa-miR-210-3p and hsa-let-7b-5p which may influence pluripotency.

Response to Cadmium in Silene vulgaris Ecotypes Is Distinctly Affected by Priming-Induced Changes in Oxidation Status of Macromolecules.

This study investigated the impact of several priming agents on metal-tolerant and sensitive Silene vulgaris ecotypes exposed to environmentally relevant cadmium dose. We analyzed how priming-induced changes in the level of lipid, protein, and DNA oxidation contribute to calamine (Cal) and non-calamine (N-Cal) ecotype response to Cd toxicity, and whether the oxidative modifications interrelate with Cd tolerance. In non-primed ecotypes, the levels of DNA and protein oxidation were similar whereas Cal Cd tolerance was manifested in reduced lipid peroxidation. In both ecotypes protective action of salicylic acid (SA) and nitric oxide (NO) priming was observed. SA stimulated growth and reduced lipid and DNA oxidation at most, while NO protected DNA from fragmentation. Priming with hydrogen peroxide reduced biomass and induced DNA oxidation. In N-Cal, priming diminished Cd accumulation and oxidative activity, whereas in Cal, it merely affected Cd uptake and induced protein carbonylation. The study showed that priming did not stimulate extra stress resistance in the tolerant ecotype but induced metabolic remodeling. In turn, the lack of adaptive tolerance made the sensitive ecotype more responsive to the benefits of the primed state. These findings could facilitate priming exploitation with a view of enhancing metallophyte and non-metallophyte suitability for phytoremediation and land revegetation.

Ectopic expression of an expansin-like B gene from wild Arachis enhances tolerance to both abiotic and biotic stresses.

Plant expansins are structural cell wall-loosening proteins implicated in several developmental processes and responses to environmental constraints and pathogen infection. To date, there is limited information about the biological function of expansins-like B (EXLBs), one of the smallest and less-studied subfamilies of plant expansins. In the present study, we conducted a functional analysis of the wild Arachis AdEXLB8 gene in transgenic tobacco (Nicotiana tabacum) plants to clarify its putative role in mediating defense responses to abiotic and biotic stresses. First, its cell wall localization was confirmed in plants expressing an AdEXLB8:eGFP fusion protein, while nanomechanical assays indicated cell wall reorganization and reassembly due to AdEXLB8 overexpression without compromising the phenotype. We further demonstrated that AdEXLB8 increased tolerance not only to isolated abiotic (drought) and biotic (Sclerotinia sclerotiorum and Meloidogyne incognita) stresses but also to their combination. The jasmonate and abscisic acid signaling pathways were clearly favored in transgenic plants, showing an activated antioxidative defense system. In addition to modifications in the biomechanical properties of the cell wall, we propose that AdEXLB8 overexpression interferes with phytohormone dynamics leading to a defense primed state, which culminates in plant defense responses against isolated and combined abiotic and biotic stresses.

Overexpression of A Biotic Stress-Inducible Pvgstu Gene Activates Early Protective Responses in Tobacco under Combined Heat and Drought.

Drought and heat stresses are major factors limiting crop growth and productivity, and their effect is more devastating when occurring concurrently. Plant glutathione transferases (GSTs) are differentially expressed in response to different stimuli, conferring tolerance to a wide range of abiotic stresses. GSTs from drought-tolerant Phaseolus vulgaris var. "Plake Megalosperma Prespon" is expected to play an important role in the response mechanisms to combined and single heat and drought stresses. Herein, we examined wild-type N. tabacum plants (cv. Basmas Xanthi) and T1 transgenic lines overexpressing the stress-induced Pvgstu3-3 and Pvgstu2-2 genes. The overexpression of Pvgstu3-3 contributed to potential thermotolerance and greater plant performance under combined stress. Significant alterations in the primary metabolism were observed in the transgenic plants between combined stress and stress-free conditions. Stress-responsive differentially expressed genes (DEGs) and transcription factors (TFs) related to photosynthesis, signal transduction, starch and sucrose metabolism, osmotic adjustment and thermotolerance, were identified under combined stress. In contrast, induction of certain DEGs and TF families under stress-free conditions indicated that transgenic plants were in a primed state. The overexpression of the Pvgstu3-3 is playing a leading role in the production of signaling molecules, induction of specific metabolites and activation of the protective mechanisms for enhanced protection against combined abiotic stresses in tobacco.

Mettl14 is required for mouse postimplantation development by facilitating epiblast maturation.

N6-methyladenosine (m6A) is the most prevalent and reversible internal modification of mammalian messenger and noncoding RNAs mediated by specific m6A writer, reader, and eraser proteins. As an m6A writer, the methyltransferase-like 3-methyltransferase-like 14 (METTL14)-Wilms tumor 1-associated protein complex dynamically regulates m6A modification and plays important roles in diverse biologic processes. However, our knowledge about the complete functions of this RNA methyltransferase complex, the contributions of each component to the methylation, and their effects on different biologic pathways are still limited. By using both in vivo and in vitro models, we here report that METTL14 is indispensable for postimplantation embryonic development by facilitating the conversion from naive to primed state of the epiblast. Depletion of Mettl14 leads to conspicuous embryonic growth retardation from embryonic d 6.5, mainly as a result of resistance to differentiation, which further leads to embryonic lethality early in gestation. Our data highlight the critical function of METTL14 as an m6A modification regulator in orchestrating early mouse embryogenesis.-Meng, T.-G., Lu, X., Guo, L., Hou, G.-M., Ma, X.-S., Li, Q.-N., Huang, L., Fan, L.-H., Zhao, Z.-H., Ou, X.-H., OuYang, Y.-C., Schatten, H., Li, L., Wang, Z.-B., Sun, Q.-Y. Mettl14 is required for mouse postimplantation development by facilitating epiblast maturation.

Primed Conversion: The New Kid on the Block for Photoconversion.

In 2015, a novel way to convert photoconvertible fluorescent proteins was reported that uses the intercept of blue and far-red light instead of traditional violet or near-UV light illumination. This Minireview describes and contrasts this distinct two-step mechanism termed primed conversion with traditional photoconversion. We provide a comprehensive overview of what is known to date about primed conversion and focus on the molecular requirements for it to take place. We provide examples of its application to axially confined photoconversion in complex tissues as well as super-resolution microscopy. Further, we describe why and when it is useful, including its advantages and disadvantages, and give an insight into potential future development in the field.

Glycans define the stemness of naïve and primed pluripotent stem cells.

Cell surface glycans are tissue-specific and developmentally regulated. They function as essential modulators in cell-cell interactions, cell-extracellular matrix interactions, and ligand-receptor interactions, binding to various ligands, including Wnt, fibroblast growth factors, and bone morphogenetic proteins. Embryonic stem (ES) cells, originally derived from the inner cell mass of blastocysts, have the essential characteristics of pluripotency and self-renewal. Recently, it has been proposed that mouse and human conventional ES cells are present in different developmental stages, namely pre-implantation blastocyst and post-implantation blastocyst stages, also called the naïve state and the primed state, respectively. They therefore require different extrinsic signals for the maintenance of self-renewal and pluripotency, and also appear to require different surface glycans. Understanding of molecular mechanisms involving glycans in self-renewal and pluripotency of ES cells is increasingly important for potential clinical applications, as well as for basic research. This review focuses on the roles of glycans in the two different states of pluripotent stem cells, namely the naïve state and the primed state, and the transition between these two states.

Label-free and non-destructive identification of naïve and primed embryonic stem cells based on differences in cellular metabolism.

Pluripotent stem cells (PSCs) exist in naïve or primed states based on their origin. For in vitro culture, these PSCs require different supplements and growth factors. However, owing to their similar phenotypic features, identifying both cell types without harming cellular functions is challenging. This study reports an electrochemical method that enables simple, label-free, and non-destructive detection of naïve embryonic stem cells (ESCs) derived from mouse ESCs, based on the differences in cellular metabolism. Two major metabolic pathways to generate adenosine triphosphate (ATP)-glycolysis and oxidative phosphorylation (OXPHOS)-were blocked, and it was found that mitochondrial energy generation is the origin of the strong electrochemical signals of naïve ESCs. The number of ESCs is quantified when mixed with primed ESCs or converted from naïve-primed switchable metastable ESCs. The mouse PSCs derived from doxycycline-inducible mouse embryonic fibroblasts (MEFs) are also sensitively identified among other cell types such as unconverted MEFs and primed PSCs. The developed sensing platform operates in a non-invasive and label-free manner. Thus, it can be useful in the development of stem cell-derived therapeutics.

Naïve-like conversion of bovine induced pluripotent stem cells from Sertoli cells.

Feeder cells are essential to derive pluripotent stem cells (PSCs). Mouse embryonic fibroblasts (MEF) are widely used as feeder to generate and culture embryonic stem cells (ESCs) and induced PSCs (iPSCs) in many species. However it may not be suitable for livestock ESCs/iPSCs due to interspecies difference. Previously we derived bovine iPSCs from bovine Sertoli cells using MEF feeder. Here we compared the effects of MEF feeder and bovine embryonic fibroblasts (BEF) feeder on the maintenance of bovine iPSC pluripotency and morphology as well their contributions to the naïve-like conversion, based on a naïve medium (NM). The results showed successful conversion of the primed bovine iPSCs to naïve-like state within 3-4 days both on MEF feeder and BEF feeder in NM (termed as MNM and BNM respectively). These naïve-like iPSCs showed normal karyotype. There were more iPSC colonies under BNM condition than MNM condition. Epigenetically, histone modification H3K4 was upregulated, while H3K27 was downregulated in the naïve-like iPSCs. We further analyzed the naïve markers and differentiation potential both in vitro and in vivo of these cells, which were all reserved throughout the maintenance. Together, bovine naïve-like iPSCs can be generated both on MEF and BEF feeder in NM condition. The BNM condition is able to sustain the pluripotency and differentiation potential of the naïve-like bovine iPSCs, and improve the conversion efficiency.

Zfp281 Inhibits the Pluripotent-to-Totipotent State Transition in Mouse Embryonic Stem Cells.

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.

Seaweed Extract-Stimulated Priming in Arabidopsis thaliana and Solanum lycopersicum.

Plant priming is an induced physiological state where plants are protected from biotic and abiotic stresses. Whether seaweed extracts promote priming is largely unknown as is the mechanism by which priming may occur. In this study, we examined the effect of a seaweed extract (SWE) on two distinct stages of plant priming (priming phase and post-challenge primed state) by characterising (i) plant gene expression responses using qRT-PCR and (ii) signal transduction responses by evaluating reactive oxygen species (ROS) production. The SWE is made from the brown algae Ascophyllum nodosum and Durvillaea potatorum. The priming phase was examined using both Arabidopsis thaliana and Solanum lycopersicum. At this stage, the SWE up-regulated key priming-related genes, such as those related to systemic acquired resistance (SAR) and activated the production of ROS. These responses were found to be temporal (lasting 3 days). The post-challenge primed state was examined using A. thaliana challenged with a root pathogen. Similarly, defence response-related genes, such as PR1 and NPR1, were up-regulated and ROS production was activated (lasting 5 days). This study found that SWE induces plant priming-like responses by (i) up-regulating genes associated with plant defence responses and (ii) increasing production of ROS associated with signalling responses.

microRNA regulation of pluripotent state transition.

microRNAs (miRNAs) play essential roles in mouse embryonic stem cells (ESCs) and early embryo development. The exact mechanism by which miRNAs regulate cell fate transition during embryo development is still not clear. Recent studies have identified and captured various pluripotent stem cells (PSCs) that share similar characteristics with cells from different stages of pre- and post-implantation embryos. These PSCs provide valuable models to understand miRNA functions in early mammalian development. In this short review, we will summarize recent work towards understanding the function and mechanism of miRNAs in regulating the transition or conversion between different pluripotent states. In addition, we will highlight unresolved questions and key future directions related to miRNAs in pluripotent state transition. Studies in these areas will further our understanding of miRNA functions in early embryo development, and may lead to practical means to control human PSCs for clinical applications in regenerative medicine.

Dppa3 is critical for Lin28a-regulated ES cells naïve-primed state conversion.

Lin28a is a pluripotent factor that promotes somatic cell reprogramming. Unlike other pluripotent factors, Lin28a expression is transient and accumulated in primed embryonic stem (ES) cells, but its exact function and mechanism in the conversion of ES cells from naïve to primed state remain unclear. Here, we present evidence for Dppa3, a protein originally known for its role in germ cell development, as a downstream target of Lin28a in naïve-primed conversion. Using rescue experiment, we demonstrate that Dppa3 functions predominantly downstream of Lin28a during naïve-primed state conversion. Higher level of Lin28a prevents let-7 maturation and results in Dnmt3a/b (target of let-7) upregulation, which in turn induces hypermethylation of the Dppa3 promoter. Dppa3 demarcates naïve versus primed pluripotency states. These results emphasize that Lin28a plays an important role during the naïve-primed state conversion of ES cells, which is partially mediated by a Lin28a-let-7-Dnmt3a/b-Dppa3 axis.

bHLH Transcription Factor Math6 Antagonizes TGF-ß Signalling in Reprogramming, Pluripotency and Early Cell Fate Decisions.

The basic helix-loop-helix (bHLH) transcription factor Math6 (Atonal homolog 8; Atoh8) plays a crucial role in a number of cellular processes during embryonic development, iron metabolism and tumorigenesis. We report here on its involvement in cellular reprogramming from fibroblasts to induced pluripotent stem cells, in the maintenance of pluripotency and in early fate decisions during murine development. Loss of Math6 disrupts mesenchymal-to-epithelial transition during reprogramming and primes pluripotent stem cells towards the mesendodermal fate. Math6 can thus be considered a regulator of reprogramming and pluripotent stem cell fate. Additionally, our results demonstrate the involvement of Math6 in SMAD-dependent TGF beta signalling. We furthermore monitor the presence of the Math6 protein during these developmental processes using a newly generated Math6Flag-tag mouse. Taken together, our results suggest that Math6 counteracts TGF beta signalling and, by this, affects the initiating step of cellular reprogramming, as well as the maintenance of pluripotency and early differentiation.

Chemically Defined Media Can Maintain Pig Pluripotency Network In Vitro.

Pig embryonic stem cells (pESCs) have been considered an important candidate for preclinical research on human therapies. However, the lack of understanding of pig pluripotent networks has hampered establishment of authentic pESCs. Here, we report that FGF2, ACTVIN, and WNT signaling are essential to sustain pig pluripotency in vitro. Newly derived pESCs were stably maintained over an extended period, and capable of forming teratomas that contained three germ layers. Transcriptome analysis showed that pESCs were developmentally similar to late epiblasts of preimplantation embryos and in terms of biological functions resembled human rather than mouse pluripotent stem cells. However, the pESCs had distinct features such as coexpression of SSEA1 and SSEA4, two active X chromosomes, and a unique transcriptional pattern. 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.

Glycans in stem cell regulation: from Drosophila tissue stem cells to mammalian pluripotent stem cells.

Cell surface glycans, which are tissue-specific and developmentally regulated, work as essential modulators in ligand-receptor interactions, binding to various signal ligands including Wnt, Hedgehog, fibroblast growth factors, epidermal growth factors, and bone morphogenetic proteins, as well as in cell-cell interactions and cell-extracellular matrix interactions. These signals are essential for the stemness and differentiation of various kinds of stem cells. In addition, the intracellular O-linked N-acetylglucosamine, a form of glycosylation found only on nuclear or cytoplasmic proteins, regulates core transcription factors of stemness and phosphorylation of downstream signal components. Therefore, various kinds of glycans regulate the stem cell status; the structures of many of which are evolutionarily conserved from Drosophila to mammals. Understanding the molecular mechanisms of glycans in stemness and differentiation is increasingly important for innovative clinical applications, as well as for basic research. This Review focuses on the roles of glycans in Drosophila tissue stem cells and mammalian pluripotent stem cells.

Dissecting the variation in transcriptional circuits between naive and primed pluripotent states.

Naive and primed pluripotent states are very similar to each other, but subtle differences exist in their maintenance and differentiation programmes. Transcription factors (TFs) play a key role towards maintaining pluripotency and cellular reprogramming. However, TF expression dynamics and regulatory mechanisms in naive and primed pluripotent states are poorly understood. Here, we performed a comprehensive transcriptional analysis of both states, which revealed a gene expression pattern in mESCs (naive state) that appear to be distinct from mEpiSCs (primed state). We screened 10 TFs essential for maintenance, self-renewal and differentiation, of which the TFs- Notch3, Meis1, Gli3 and Srf can act as novel markers distinguishing the two states. Furthermore, a detailed bioinformatic analysis (involving these TFs) elucidated essential transcriptional circuits between the naive and primed pluripotent states.

Comprehensive Mapping of Pluripotent Stem Cell Metabolism Using Dynamic Genome-Scale Network Modeling.

Metabolism is an emerging stem cell hallmark tied to cell fate, pluripotency, and self-renewal, yet systems-level understanding of stem cell metabolism has been limited by the lack of genome-scale network models. Here, we develop a systems approach to integrate time-course metabolomics data with a computational model of metabolism to analyze the metabolic state of naive and primed murine pluripotent stem cells. Using this approach, we find that one-carbon metabolism involving phosphoglycerate dehydrogenase, folate synthesis, and nucleotide synthesis is a key pathway that differs between the two states, resulting in differential sensitivity to anti-folates. The model also predicts that the pluripotency factor Lin28 regulates this one-carbon metabolic pathway, which we validate using metabolomics data from Lin28-deficient cells. Moreover, we identify and validate metabolic reactions related to S-adenosyl-methionine production that can differentially impact histone methylation in naive and primed cells. Our network-based approach provides a framework for characterizing metabolic changes influencing pluripotency and cell fate.

Calcium signaling in human pluripotent stem cells.

Human pluripotent stem cells provide new tools for developmental and pharmacological studies as well as for regenerative medicine applications. Calcium homeostasis and ligand-dependent calcium signaling are key components of major cellular responses, including cell proliferation, differentiation or apoptosis. Interestingly, these phenomena have not been characterized in detail as yet in pluripotent human cell sates. Here we review the methods applicable for studying both short- and long-term calcium responses, focusing on the expression of fluorescent calcium indicator proteins and imaging methods as applied in pluripotent human stem cells. We discuss the potential regulatory pathways involving calcium responses in hPS cells and compare these to the implicated pathways in mouse PS cells. A recent development in the stem cell field is the recognition of so called "naïve" states, resembling the earliest potential forms of stem cells during development, as well as the "fuzzy" stem cells, which may be alternative forms of pluripotent cell types, therefore we also discuss the potential role of calcium homeostasis in these PS cell types.

p53 and p73 Regulate Apoptosis but Not Cell-Cycle Progression in Mouse Embryonic Stem Cells upon DNA Damage and Differentiation.

Embryonic stem cells (ESCs) are fast proliferating cells capable of differentiating into all somatic cell types. In somatic cells, it is well documented that p53 is rapidly activated upon DNA damage to arrest the cell cycle and induce apoptosis. In mouse ESCs, p53 can also be functionally activated, but the precise biological consequences are not well characterized. Here, we demonstrated that doxorubicin treatment initially led to cell-cycle arrest at G2/M in ESCs, followed by the occurrence of massive apoptosis. Neither p53 nor its target gene p73 was required for G2/M arrest. Instead, p53 and p73 were fully responsible for apoptosis. p53 and p73 were also required for differentiation-induced apoptosis in mouse ESCs. In addition, doxorubicin treatment induced the expression of retinoblastoma protein in a p53-dependent manner. Therefore, both p53 and p73 are critical in apoptosis induced by DNA damage and differentiation.