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• W68243353 endingPage "1473" @default.
• W68243353 startingPage "1473" @default.
• W68243353 abstract "Abstract Abstract 1473 Poster Board I-496 Mouse embryonic stem cells (mESCs) are unique in that they give rise to every cell type of the body. Little is known about stimuli that promote mESC differentiation and proliferation. We hypothesized that TLRs are expressed and functional, and when activated by its ligand will influence survival and proliferation of mESCs in the presence of Leukemia Inhibitory Factor (LIF). This study evaluated three mESC lines, R1, CGR8, and E14 to first determine if they express Toll Like Receptors (TLRs) at mRNA and protein levels. Next, we evaluated if the TLR ligands would induce or modulate mESC proliferation and survival of the mESCs in the presence of LIF. We then assessed the E14 mESC line to determine if the mESCs expressed MyD88 an adaptor protein molecule, known to be involved in the TLR pathway and if the TLR ligands would cause inhibitor of kinase kinase alpha/ beta (IKKα/β) phosphorylation and nuclear factor-kappa beta (NF-κβ) nuclear translocation, and cytokine production. In this study we found expression of TLRs 1, 2, 3, 5 and 6 at the mRNA level, but no mRNA expression of TLRs 4, 7, 8 and 9. We also confirmed some of these results by flow analysis. Toll Like Receptor 2 (TLR-2), but not Toll Like Receptor 4 (TLR-4), is expressed on the three mESC lines. Therefore we focused our studies on TLR-2, specifically. Pam3Cys, a synthetic triacyl-lipoprotein and a TLR-2 ligand induced a significant increase in mESC numbers on Day 3 compared to controls and also at Day 4 and Day 5 when compared to controls. Pam3Cys (10ug/ml) also enhanced the survival of mESC colony forming cells subjected to serum withdrawal and then delayed addition of serum. Next we found that E14 mESCs express molecules involved in the TLR Pathway. MyD88 was expressed in mESCs and IKKα/β phosphorylation was enhanced after 15 minute by TLR-2 ligand activation. We found a significant increase of NF-κβ nuclear translocation upon activation by Pam3Cys after 30 minutes which continued for up to an hour. Densitometry analysis of the nuclear extracts from three separate experiments shows a significant 2 fold increase in NF-κβ in the nucleus compared to control mESC nuclear extracts. Since TLR activation of leukocytes enhances cytokine production, and our group has published that mESCs produce cytokines, we studied the effect of Pam3Cys on release of cytokine from our mESC line. Cytokine release in mESCs by TLR-ligand activation was assessed by ELISA, but TLR-2 ligand stimulation did not affect cytokine release of IL-6, TNF-α, or INF-β. We found that there were no significant changes in expression of mESCs markers Oct4, KLF-4, Sox 2, and SSEA1 when compared to cells not activated by Pam3Cys. Thus the mESCs remained in a pluripotent state in the presence of LIF after activation with the TLR-2 ligand. These results demonstrate that mESCs can respond to microbial products, and TLR-2 activation enhances proliferation and survival of the mESCs. This finding expands the role of TLRs and has implications for a better understanding of the responsiveness of embryonic stem cells to certain microbial agents. Disclosures: No relevant conflicts of interest to declare." @default.
• W68243353 created "2016-06-24" @default.
• W68243353 creator A5078228694 @default.
• W68243353 date "2009-11-20" @default.
• W68243353 modified "2023-10-01" @default.
• W68243353 title "Mouse Embryonic Stem Cells Express Functional Toll Like Receptor 2." @default.
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