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Title

Zebularine regulates early stages of mESC differentiation: effect on cardiac commitment

AuthorsHorrillo, Angélica ; Pezzolla, Daniela ; Fraga, Mario F.; Aguilera, Yolanda ; Salguero-Aranda, Carmen ; Tejedo Huamán, Juan Rigoberto ; Martín, Franz ; Bedoya Bergua, Francisco Javier ; Soria Escoms, Bernat ; Hmadcha, Abdelkrim
KeywordsEmbryonic stem cells
Mesoderm
Methylation
Zebularine
AzadC
Nitric oxide
Issue Date4-Apr-2013
PublisherNature Publishing Group
CitationCell Death and Disease 4: e570 (2013)
AbstractLineage 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 50-aza-20-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 lM 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. © 2013 Macmillan Publishers Limited All rights reserved.
Publisher version (URL)http://dx.doi.org/10.1038/cddis.2013.88
URIhttp://hdl.handle.net/10261/122765
DOI10.1038/cddis.2013.88
Identifiersdoi: 10.1038/cddis.2013.88
issn: 2041-4889
Appears in Collections:(CABIMER) Artículos
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