Balancing Safety and Developmental Potency: Assessing the Effect of Antioxidants and TET on Genomic Imprinting Stability.

Abstract

Developmental potency represents the ability of undifferentiated cells to undergo transformation into differentiated cells, with specialised functions. The inner cell mass (ICM) of a blastocyst represents the most developmentally potent cells of the body. The ICM, along with the naïve embryonic stem cells derived from it, possess remarkably low levels of global DNA methylation; a trait that is intrinsic to their pluripotent phenotype and when artificially induced can help re-program differentiated cells back into a naïve state. Ten-eleven translocation (TET) enzymes actively remove DNA methylation in a Fe2+-dependent fashion. By assisting Fe2+ recycling, the antioxidant ascorbate has been shown to increase TET enzymatic activity and enhance reprogramming of differentiated cells to the naïve state. However, it is currently unclear if TET activation via ascorbate, or other antioxidants, can cause unwanted demethylation at imprint control regions; regulatory elements that control parent-of-origin specific gene expression. Loss of methylation at these regulatory groups results in the biallelic expression of the genes under their control. This phenomenon has been implicated in the formation of a range of cancers, as well as Beckwith-Wiedemann syndrome, a congenital overgrowth disorder. In this project, I aimed to test what effect the antioxidants ascorbate and hydroquinone have on the stability of imprint control regions through the activation of TET proteins, and in doing so, assess the safety and practicality of using naïve embryonic stem cells for future medical applications. I grew several naïve embryonic stem cell lines, including a TET triple knockout line with inducible TET expression, in varying concentrations of ascorbate or hydroquinone. Bisulfite amplicon sequencing and florescence activated cell sorting techniques were used to assess methylation patterns at the KCNQ1ot1 imprinted loci in the presence or absence of increased TET activity. While the study has been hampered by technical difficulties (i.e. clonal amplification was detected, particularly in TET knockout cell lines), I have shown that ascorbate causes significant demethylation of the KCNQ1ot1 imprinted region at 25 ng/μL of ascorbate. While other concentrations, (12.5 ng/μL and 50 ng/μL of ascorbate) were not statistically significant there appeared to be a trend of decreasing methylation which may have become more obvious had the cells been cultured with ascorbate for longer. Interestingly this effect was not seen at 100 ng/μL of ascorbate, indicating there is an optimal concentration range for TET-induced demethylation at the KCNQ1ot1 locus. In contrast, hydroquinone had no significant effect. Ultimately, these results add to the current literature describing the effects of different culture media constituents on naïve embryonic stem cells. This may allow for the rational design of culture media that can improve both the efficiency and safety of naïve embryonic stem cells and embryos used in regenerative medicine, mammalian transgenics and IVF.