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Cell Journal، جلد ۲۰، شماره ۳، صفحات ۳۶۱-۳۶۸
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عنوان انگلیسی Temporal Gene Expression and DNA Methylation during Embryonic Stem Cell Derivation
چکیده انگلیسی مقاله Objective: Dual inhibition of mitogen-activated protein kinase (MAPK) kinase (also known as MEK) and transforming growth factor β (TGFβ) type I receptors by PD0325901 and SB431542, known as R2i has been introduced as a highly efficient approach to the generation of mouse embryonic stem cells . In the present study, we investigated the molecular mechanisms underlying ESC derivation in the R2i condition. Materials and methods: Zona-free whole E3.5 blastocysts were seeded on MEF feeder cells in both R2i and serum conventional media. The isolated inner cell mass (ICM), ESCs and the ICM-outgrowths were collected on days 3, 5 and 7 post-blastocyst culture for qRT-PCR analysis as well as to assess the DNA methylation status at the time points during the transition from ICM to ESC. Results: qRT-PCR revealed a significantly higher expression of the pluripotency-related genes (Oct4, Nanog, Sox2, Rex1, Dppa3, Tcf3, Utf1, Nodal, Dax1, Sall4 and β-Catenin) and lower expression of early differentiation genes (Gata6, Lefty2 and Cdx2) in R2i condition compared to the serum condition. Moreover, the upstream region of Oct4 and Nanog showed a progressive increase in methylation levels in the upstream regions of the genes following in R2i or serum conditions, followed by a decrease of DNA methylation in ESCs obtained under R2i. However, the methylation level of ICM outgrowths in the serum condition was much higher than R2i, at levels that could have a repressive effect and therefore explain the absence of expression of these two genes in the serum condition. Conclusion: We can conclude from all data obtained through the inhibition of the MEK and TGFβ signaling pathways that components of the culture medium in the first 5 days of ESC generation can affect the establishment of the ground-state of pluripotency.
کلیدواژه‌های انگلیسی مقاله
نویسندگان مقاله | Azam Samadian


| Mahdi Hesaraki


| Sepideh Mollamohamadi


| Behrouz Asgari


| Mehdi Totonchi


| Hossein Baharvand


نشانی اینترنتی http://celljournal.org/journal/article/abstract/5482
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