Project description:Leukemia Inhibitory Factor (LIF) plays an essential role in the maintenance of pluripotency of mouse embryonic stem cells (mESCs). LIF withdrawal induces mESC differentiation. To define noval pluripotent factors downstream of LIF signaling, cDNA microarray was used and seveal well-known pluripotent genes were found to respond to LIF withdrawal, including Klf4, Esrrb, Tbx3, and Prdm14.
Project description:Leukemia Inhibitory Factor (LIF) plays an essential role in the maintenance of pluripotency of mouse embryonic stem cells (mESCs). LIF withdrawal induces mESC differentiation. To define noval pluripotent factors downstream of LIF signaling, cDNA microarray was used and seveal well-known pluripotent genes were found to respond to LIF withdrawal, including Klf4, Esrrb, Tbx3, and Prdm14. mESCs were cultured in presence or absence of LIF for two days and RNAs extracted from these cells were subjected to microarray analysis
Project description:Self-renewal and pluripotency are hallmarks of embryonic stem cells (ESCs). However, signaling pathways triggering their transition from self-renewal to differentiation are not well defined. Here, we report that the calcineurin-NFAT signaling pathway is both necessary and sufficient to switch ESCs from an undifferentiated state to early differentiation.To gain an insight into the function of calcinerin-NFAT signaling in ESC lineage commitment, we examined the repertoire of genes changed after LIF withdrawal alone, LIF withdrawal in the presence of cyclosporine A (CsA), a specific inhibitor of calcinerin and forced expression of active forms of calcineurin (deltaCnA) in the presence of LIF,and identified distinct classes of up and down regulated genes by calcineurin-NFAT signaling in mouse embryonic stem cells. Total RNA obtained from CGR8 ES cells under the following conditions: withdrawal of LIF for 0, 1 and 3 days; withdrawal of LIF for 0, 1 and 3 days in the presence of CsA, and induced expression of deltaCnA by withdrawal of Tc for 0, 1 and 4 days in ideltaCnAES cells.Hybridized to Illumina Sentrix Mouse-6 v1.1 Beadhips
Project description:Self-renewal and pluripotency are hallmarks of embryonic stem cells (ESCs). However, signaling pathways triggering their transition from self-renewal to differentiation are not well defined. Here, we report that the calcineurin-NFAT signaling pathway is both necessary and sufficient to switch ESCs from an undifferentiated state to early differentiation.To gain an insight into the function of calcinerin-NFAT signaling in ESC lineage commitment, we examined the repertoire of genes changed after LIF withdrawal alone, LIF withdrawal in the presence of cyclosporine A (CsA), a specific inhibitor of calcinerin and forced expression of active forms of calcineurin (deltaCnA) in the presence of LIF,and identified distinct classes of up and down regulated genes by calcineurin-NFAT signaling in mouse embryonic stem cells.
Project description:An NFkB target gene array (Panomics, Inc., Redwood City, CA) was performed to profile the expression pattern of 107 NFkB-regulated genes in mouse embryonic stem cells. A small scale microarray was carried out using NFkB target gene array kit (Panomics, Inc., Redwood City, CA). A long sense-strand oligonucleotide for each of the 107 human genes that have been previously shown to be regulated by NFkB signaling pathway was spotted in duplicate on the nitrocellulose membrane. Biotinylated DNA was spotted along the right and bottom sides of the array membrane as control. The arrays were performed according to the manufacturer’s instructions. Keywords: embryonic stem cell differentiation
Project description:Transcriptomic analysis of mouse embryonic stem cells (mESC) maintained in the pluripotent state or allowed to differentiate for 3 or 7 days by leukaemia inhibitory factor (LIF) withdrawal.
Project description:Mouse embryonic stem (ES) cells remain pluripotent in vitro when grown in presence of Leukaemia Inhibitory Factor (LIF). LIF starvation leads to apoptosis of some of the ES-derived differentiated cells, together with p38a MAP kinase activation. Apoptosis, but not morphological cell differentiation, is blocked by a p38 inhibitor, PD 169316. To further understand the mechanism of action of this compound, we have identified its specific targets by microarray studies. We report on the global expression profiles of genes expressed at three days upon LIF withdrawal (d3) compared to pluripotent cells and of genes whose expression is modulated at d3 under anti-apoptotic conditions. We showed that at d3 without LIF cells express, earlier than anticipated, specialized cell markers and that when the apoptotic process was impaired, expression of differentiation markers was altered. In addition, functional tests revealed properties of anti-apoptotic proteins not to alter cell pluripotency and a novel role for metallothionein 1 gene which prevents apoptosis of early differentiated cells. Keywords: mouse ES cells; apoptosis; differentiation; p38; PD169316; Ca++ binding proteins; Metallothionein; stress-response Keywords: ordered
Project description:The decline in stem cell function leads to impairments in tissue homeostasis but genetic factors that control differentiation and de-differentiation of stem cells in the context of tissue homeostasis remain to be delineated. Here we show that Tnfaip2 (a target gene of TNFa/NFkB signaling) has an essential role for the differentiation of pluripotent, embryonic stem cells (ESCs). Knockdown of the planarian pseudo-orthologue, Smed-exoc3, impairs pluripotent stem cell differentiation, tissue homeostasis and regeneration in vivo. The study shows that Tnfaip2 deletion impairs changes in lipid metabolism that drive differentiation induction of ESCs. The application of palmitic acid (PA, the most abundant saturated fatty acid in mammalian cells) and palmitoylcarnitine (a mitochondrial carrier of PA) fully restores the differentiation of ESCs as well as the differentiation of pluripotent stem cells and organ maintenance in Smed-exoc3-depleted planarians. Together, these results identify a novel pathway downstream of TNFa/NFkB signaling, which is essential for exit from pluripotency by mediating changes in lipid metabolism.
Project description:Mouse embryonic stem (ES) cells remain pluripotent in vitro when grown in presence of Leukaemia Inhibitory Factor (LIF). LIF starvation leads to apoptosis of some of the ES-derived differentiated cells, together with p38a MAP kinase activation. Apoptosis, but not morphological cell differentiation, is blocked by a p38 inhibitor, PD 169316. To further understand the mechanism of action of this compound, we have identified its specific targets by microarray studies. We report on the global expression profiles of genes expressed at three days upon LIF withdrawal (d3) compared to pluripotent cells and of genes whose expression is modulated at d3 under anti-apoptotic conditions. We showed that at d3 without LIF cells express, earlier than anticipated, specialized cell markers and that when the apoptotic process was impaired, expression of differentiation markers was altered. In addition, functional tests revealed properties of anti-apoptotic proteins not to alter cell pluripotency and a novel role for metallothionein 1 gene which prevents apoptosis of early differentiated cells.