Abstract
Nitric oxide (NO) synthesis is a late event during differentiation of mouse embryonic stem cells (mESC) and occurs after release from serum and leukemia inhibitory factor (LIF). Here we show that after release from pluripotency, a subpopulation of mESC, kept in the naive state by 2i/LIF, expresses endothelial nitric oxide synthase (eNOS) and endogenously synthesizes NO. This eNOS/NO-positive subpopulation (ESNO+) expresses mesendodermal markers and is more efficient in the generation of cardiovascular precursors than eNOS/NO-negative cells. Mechanistically, production of endogenous NO triggers rapid Hdac2 S-nitrosylation, which reduces association of Hdac2 with the transcriptional repression factor Zeb1, allowing mesendodermal gene expression. In conclusion, our results suggest that the interaction between Zeb1, Hdac2, and eNOS is required for early mesendodermal differentiation of naive mESC.
Highlights
Nitric oxide (NO) synthesis is a late event during differentiation of mouse embryonic stem cells and occurs after release from serum and leukemia inhibitory factor (LIF)
To compare the biological responses to NO of mouse embryonic stem cells (mESC) grown in standard medium (SM) vs. ground state-like” (GS), cells were released for 24 h from inhibitors and cultured in complete medium supplemented with 10% fetal bovine serum in the absence or presence of the NO donor diethylenetriamine NONOate[32] (DM plus DETA/NO, NO)
In SM, miR-200b, miR-200a, and miR-429 were induced after LIF withdrawal (DM), an effect enhanced in NO (Supplementary Fig. 1c, middle upper graph)[28]
Summary
Nitric oxide (NO) synthesis is a late event during differentiation of mouse embryonic stem cells (mESC) and occurs after release from serum and leukemia inhibitory factor (LIF). A body of literature, established NO as an essential factor for cardiovascular precursor generation during mESC cardiovascular lineage commitment[3,4,5] This effect seems to depend on experimental conditions and, at least in one report, it has been described that low doses of NO may repress differentiation[6]. Of note, all these observations were made by adding exogenous sources of NO to the mESC medium or expressing a wild-type eNOS in cells cultured in the presence of leukemia inhibitory factor (LIF)[2,4,5,7]. Zeb[1] genetic inactivation determined complex phenotypes affecting the development of ectodermal and mesendodermal structures[24,25,26,27]
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