Epigenetically Reprogramming of Human Embryonic Stem Cells By 3-Deazaneplanocin A and Sodium Butyrate

Abstract

Infertility affects about 6.1 million women ages 15-44 in the United States.  The leading cause of infertility in women is quantitative and qualitative defects in human germ-cell development. Human embryonic stem cell (hESC) lines are derived from the inner cell mass (ICM) of developing blastocysts and have a broad clinical potential, and they can be used to obtain germ cells. hESCs have been classified in to three classes based on their epigenetic state.  The goal of this project is to epigenetically reprogram Class II and Class III cell lines to Class I (naïve state), and to in vitro differentiate potent hESCs to primordial germ cells (PGCs). Recent evidence suggests that 3-deazaneplanocin A (DZNep) is a global histone methylation inhibitor which selectively inhibits trimethylation of lysine 27 on histone H3K27, and it is an epigenetic therapeutic for cancer. The characteristics of DZNep lead us to hypothesize that it is a good candidate to epigenetically reprogram hESC cells to the Class I. Also, we used sodium butyrate, shown in previous studies to up-regulate the expression of germ cell specific markers. We used these two drugs to produce epigenetically stable hESC lines. X-Chromosome inactivation has been used as a tool to follow the reprogramming process. We have used immunostaining and western blot as methods to follow this re-programming qualitatively and quantitatively. The preliminary results showed that hESC cells treated with these two drugs showed dramatic tendency to lose H3K27me3 marker.