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MicroRNAs (miRNAs), a group of noncoding RNAs, function as post-transcriptional gene regulators and control the establishment, self-renewal and differentiation of stem cells. Vitamin C has been recognized as a reprogramming enhancer because of its ability to induce a blastocyst-like state in embryonic stem cells (ESCs). However, knowledge on the regulation of miRNAs by vitamin C in ESCs is limited. In this study, we found that vitamin C induced miRNA expression, particularly of ESC-specific miRNAs. Moreover, vitamin C maintained the miRNA expression of the Dlk1-Dio3 imprinting region. The miRNAs in this region contain identical seed sequences, which target a class of genes, including Kdm6b, Klf13, and Sox6, and are mainly related to cell differentiation and development. These genes were significantly downregulated by vitamin C. Notably, miR-143 promoted self-renewal of mouse ESCs and suppressed expression of the de novo methyltransferase gene Dnmt3a. Knockdown of miR-143 by use of its inhibitor counteracted the vitamin C-induced reduction in Dnmt3a expression, showing that vitamin C repressed Dnmt3a expression via miR-143. Vitamin C also promoted DNA demethylation, including of pluripotency gene promoters (Tbx3, Tcl1, and Esrrb) and ESC-specific miRNA promoters (miR-290-295 and miR-17-92 clusters), and DNA hydroxymethylation, including of the intergenic differentially methylated region of the Dlk1-Dio3 region. These results strongly suggested that vitamin C promoted widespread DNA demethylation in gene promoters by modulating epigenetic modifiers, including Dnmt3a, which activated pluripotency genes and ESC-specific miRNAs. Then, differentiation and development genes were repressed by ESC-enriched miRNAs, which maintained the stem cell state., (© 2014 FEBS.)

Vitamin C has recently received attention because of its ability to improve induced pluripotent stem cells (iPSCs) generation [1-3] and maintain a blastocyst-like state in ES cells [4]. However, the underlying mechanisms are not fully understood. In this study, we found that vitamin C maintained the morphology of mouse embryonic stem cell (mESC) colonies and inhibited mESC differentiation. Gene expression profiling revealed that the genes down-regulated by vitamin C were grouped in the regulation of differentiation and development, while most of the up-regulated genes were enriched in the regulation of transcription involving numerous pluripotency factors, which was further confirmed by real time quantitative PCR. For the key pluripotency factor Nanog, vitamin C increased its promoter activity and protein level. In addition, pathway screening indicated that vitamin C may affect various signaling pathways. Our study provides new insights into vitamin C-mediated pluripotency maintenance of mESCs., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Embryonic stem cells (ESCs) can proliferate indefinitely in vitro and differentiate into cells of all three germ layers. These unique properties make them exceptionally valuable for drug discovery and regenerative medicine. However, the practical application of ESCs is limited because it is difficult to derive and culture ESCs. It has been demonstrated that CHIR99021 (CHIR) promotes self-renewal and enhances the derivation efficiency of mouse (m)ESCs. However, the downstream targets of CHIR are not fully understood. In this study, we identified CHIR-regulated genes in mESCs using microarray analysis. Our microarray data demonstrated that CHIR not only influenced the Wnt/β-catenin pathway by stabilizing β-catenin, but also modulated several other pluripotency-related signaling pathways such as TGF-β, Notch and MAPK signaling pathways. More detailed analysis demonstrated that CHIR inhibited Nodal signaling, while activating bone morphogenetic protein signaling in mESCs. In addition, we found that pluripotency-maintaining transcription factors were up-regulated by CHIR, while several developmental-related genes were down-regulated. Furthermore, we found that CHIR altered the expression of epigenetic regulatory genes and long intergenic non-coding RNAs. Quantitative real-time PCR results were consistent with microarray data, suggesting that CHIR alters the expression pattern of protein-encoding genes (especially transcription factors), epigenetic regulatory genes and non-coding RNAs to establish a relatively stable pluripotency-maintaining network., (© 2013 Elsevier Inc. All rights reserved.)

Background/aims: [corrected] Embryonic stem cells (ES cells) have the capacity to propagate indefinitely, maintain pluripotency, and differentiate into any cell type under defined conditions. As a result, they are considered to be the best model system for research into early embryonic development. AICA ribonucleotide (AICAR) is an activator of AMP-activated protein kinase (AMPK) that is thought to affect ES cell function, but its role in ES cell fate decision is unclear., Methods: In this study, we performed microarray analysis to investigate AICAR downstream targets and further understand its effect on ES cells., Results: Our microarray data demonstrated that AICAR can significantly up-regulate pluripotency-associated genes and down-regulate differentiation-associated transcription factors. Although AICAR cannot maintain ES cell identity without LIF, it can antagonize the action of RA-induced differentiation. Using those differentially expressed genes identified, we performed gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis with the Database for Annotation, Visualization and Integrated Discovery (DAVID) online system. AICAR was not only shown to influence the AMPK pathway, but also act on other signaling pathways such as BMP, MAPK and TGF-β, to maintain the stemness of J1 ES cells. Furthermore, AICAR modulated ES cell epigenetic modification by altering the expression of epigenetic-associated proteins, including Dnmt3a, Dnmt3b, Smarca2, Mbd3, and Arid1a, or through regulating the transcription of long intervening non-coding RNA (lincRNA)., Conclusion: Taken together, our work suggests that AICAR is capable of maintaining ES cell self-renewal and pluripotency, which could be useful in future medical treatment., (© 2013 S. Karger AG, Basel.)

Understanding the mechanisms that regulate pluripotency of embryonic stem cells (ESCs) is important to ensure their safe clinical use. CHIR99021 (CHIR)-induced activation of Wnt/β-catenin signaling promotes self-renewal in mouse ESCs (mESCs). β-catenin functions individually or cooperates with transcription factors to activate stemness factors such as c-Myc, Esrrb, Pou5f1, and Nanog. However the relationship between the core pluripotent factor, Kruppel-like factor 4 (also known as GKLF or EZF) and Wnt/β-catenin signaling, remains ambiguous in J1 mESCs. DNA microarray analysis revealed that CHIR-treatment promoted pluripotency-maintaining transcription factors and repressed germ layer specification markers. CHIR also promoted genes related to the development of extracellular regions and the plasma membrane to maintain pluripotency of J1 mESCs. Among the CHIR-regulated genes, Klf4 has not been reported previously. We identified a novel cis element in the Klf4 gene that was activated by β-catenin in J1 mESCs. We determined that β-catenin interacted with this cis element, identifying Klf4 as a β-catenin target gene in this context. Moreover, several microRNAs that targeted the 3′-UTR of Klf4 mRNA were identified, with miR-7a being down-regulated by CHIR in a β-catenin-independent manner in J1 mESCs. These data collectively suggest that CHIR enhances Klf4 expression by repressing miR-7a expression or canonical Wnt pathway activation. [ABSTRACT FROM AUTHOR]

ES cells can propagate indefinitely, maintain self-renewal, and differentiate into almost any cell type of the body. These properties make them valuable in the research of embryonic development, regenerative medicine, and organ transplantation. MicroRNAs (miRNAs) are considered to have essential functions in the maintenance and differentiation of embryonic stem cells (ES cells). It was reported that, strong external stimuli, such as a transient low-pH and hypoxia stress, were conducive to the formation of induced pluripotent stem cells (iPS cells). AICA ribonucleotide (AICAR) is an AMP-activated protein kinase activator, which can let cells in the state of energy stress. We have demonstrated that AICAR can maintain the pluripotency of J1 mouse ES cells through modulating protein expression in our previous research, but its effects on ES cell miRNA expression remain unknown. In this study, we conducted small RNA high-throughput sequencing to investigate AICAR influence on J1 mouse ES cells by comparing the miRNA expression patterns of the AICAR-treated cells and those without treatment. The result showed that AICAR can significantly modulate the expression of multiple miRNAs, including those have crucial functions in ES cell development. Some differentially expressed miRNAs were selected and confirmed by real-time PCR. For the differently expressed miRNAs identified, further study was conducted regarding the pluripotency and differentiation associated miRNAs with their targets. Moreover, miR-134 was significantly down-regulated after AICAR treatment, and this was suggested to be directly associated with the up-regulated pluripotency markers, Nanog and Sox2. Lastly, Myc was significantly down-regulated after AICAR treatment; therefore, we predicted miRNAs that may target Myc and identified that AICAR induced up-regulation of miR-34a, 34b, and 34c can repress Myc expression in J1 mouse ES cells. Taken together, our study provide a new mechanism for AICAR in ES cells pluripotency maintenance and give insight for its usage in iPS cells generation. [ABSTRACT FROM AUTHOR]

Background: Colony morphology of embryonic stem (ES) cells contributes to the maintenance of undifferentiated ES cells. Small molecule 3,4-dihydropy-rimido[4,5-d]pyrimidine (SC1), an inhibitor of ERK1- and RasGAP-dependent signaling pathways, can maintain the compact colony morphology of ES cells. However, information on the influence of SC1 on cell morphological change remains lacking. Methods: In this study, mouse ES cells J1 and embryonic carcinoma (EC) cells F9 were cultured in SC1-containing medium to determine the effect of SC1 on cell morphology. Results: SC1 promotes a more compact morphology of J1 mouse ES cells and induces colony growth of F9 EC cells. Furthermore, the cell adhesion protein E-cadherin is a downstream target of SC1, and E-cadherin is critical for SC1-mediated colony growth of F9 EC cells. Conclusions: SC1 maintains and induces compact colony morphology of pluripotent cells, and its downstream target, E-cadherin, is involved in the colony phenotype of F9 EC cells. These results explored the potential role of SC1 in morphological change and gene expression in pluripotent cells. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]