Past Issue

Volume 20, Number 3, Autumn 2018, Serial Number: 79, Pages: 361-368

Temporal Gene Expression and DNA Methylation during Embryonic Stem Cell Derivation


Azam Samadian, M.Sc, 1, #, Mahdi Hesaraki, M.Sc, 1, #, Sepideh Mollamohammadi, M.Sc, 1, Behrouz Asgari, M.Sc, 1, Mehdi Totonchi, Ph.D., 1, 2, *, Hossein Baharvand, Ph.D., 1, 3, *,
Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran
Department of Developmental Biology, University of Science and Culture, Tehran, Iran
*Corresponding Address: P.O.Box: 16635-141 Department of Stem Cells and Developmental Biology Cell Science Research Center Royan Institute for Stem Cell Biology and Technology ACECR Tehran Iran Emails:m.totonchi@royaninstitue.org,baharvand@royaninstitute.org

The first two authors equally contributed to this article.

Abstract

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 (ESC). In the present study, we investigated the molecular mechanisms underlying ESC derivation in the R2i condition.

Materials and Methods

In this experimental study, zona-free whole E3.5 blastocysts were seeded on mouse embryonic fibroblast (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 quantitative real time- polymerase chain reaction (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

Our investigation revealed that generation of ESCs in the ground-state of pluripotency could be achieved by inhibiting the MEK and TGF-β signaling pathways in the first 5 days of ESC derivation.