Horrillo, APezzolla, DFraga, M FAguilera, YSalguero-Aranda, CTejedo, J RMartin, FBedoya, F JSoria, BHmadcha, A2016-07-052016-07-052013-04-04Horrillo A, Pezzolla D, Fraga MF, Aguilera Y, Salguero-Aranda C, Tejedo JR, et al. Zebularine regulates early stages of mESC differentiation: effect on cardiac commitment. Cell Death Dis; 4:e570http://hdl.handle.net/10668/2254Journal Article; Research Support, Non-U.S. Gov't;Lineage commitment during embryonic stem cell (ESC) differentiation is controlled not only by a gamut of transcription factors but also by epigenetic events, mainly histone deacetylation and promoter DNA methylation. The DNA demethylation agent 5'-aza-2'-deoxycytidine (AzadC) has been widely described as an effective promoter of cardiomyogenic differentiation in various stem cell types. However, its toxicity and instability complicate its use. Therefore, the purpose of this study was to examine the effects of zebularine (1-(β-D-ribofuranosyl)-1,2-dihydropyrimidin-2-1), a stable and non-toxic DNA cytosine methylation inhibitor, on mouse ESC (mESC) differentiation. Herein, we report that treating embryoid bodies, generated from mESCs, with 30 μM zebularine for 7 days led to greater cell differentiation and induced the expression of several cardiac-specific markers that were detected using reverse transcription-polymerase chain reaction (RT-PCR), real-time PCR, immunostaining and flow cytometry. Zebularine enhanced the expression of cardiac markers and the appearance of beating cells that responded to cardiac drugs, including ion channel blockers (diltiazem) and β-adrenergic stimulators (isoproterenol). Gene promoter methylation status was assessed using methylation-specific PCR (MSP) and validated by bisulfite sequencing analysis. Global gene expression profiling using microarrays showed that zebularine-differentiated cells are distinct from control ESCs. Pathway analysis revealed an enhancement of cellular processes such as embryonic development, cardiovascular system development and function. In addition, the whole-cell proteins exhibited different profiles as analyzed by two-dimensional differential-in-gel-electrophoresis. Our results indicate that zebularine regulates mesodermal differentiation of mESCs, controls promoter methylation of crucial cardiac genes and may help to improve cardiomyogenic differentiation.enEmbryonic stem cellsMethylationMesodermZebularineAzadCNitric oxideCélulas madre embrionariaMetilaciónMesodermoLínea celularMedical Subject Headings::Chemicals and Drugs::Biological Factors::BiomarkersMedical Subject Headings::Phenomena and Processes::Cell Physiological Phenomena::Cell Physiological Processes::Cell DifferentiationMedical Subject Headings::Anatomy::Cells::Cells, Cultured::Cell LineMedical Subject Headings::Chemicals and Drugs::Nucleic Acids, Nucleotides, and Nucleosides::Nucleosides::Ribonucleosides::CytidineMedical Subject Headings::Phenomena and Processes::Genetic Phenomena::Genetic Processes::DNA MethylationMedical Subject Headings::Anatomy::Cells::Stem Cells::Embryonic Stem Cells::Embryoid BodiesMedical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Genetic Techniques::Gene Expression ProfilingMedical Subject Headings::Phenomena and Processes::Genetic Phenomena::Genetic Processes::Gene Expression Regulation::Gene Expression Regulation, DevelopmentalMedical Subject Headings::Organisms::Eukaryota::Animals::Chordata::Vertebrates::Mammals::Primates::Haplorhini::Catarrhini::Hominidae::HumansMedical Subject Headings::Phenomena and Processes::Metabolic Phenomena::Metabolism::Metabolic Networks and PathwaysMedical Subject Headings::Organisms::Eukaryota::Animals::Chordata::Vertebrates::Mammals::Rodentia::Muridae::Murinae::MiceMedical Subject Headings::Anatomy::Cells::Muscle Cells::Myocytes, CardiacMedical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Genetic Techniques::Nucleic Acid Amplification Techniques::Polymerase Chain Reaction::Real-Time Polymerase Chain Reactionresearch article23559004open access10.1038/cddis.2013.882041-4889PMC3668624