Vitamin C programming of self-renewal and pluripotency in rat embryonic stem cells

Pluripotent embryonic stem cells (ESCs) can be expanded indefinitely under self-renewing conditions, but when switched to a permissive environment can form any differentiated cell types, including germ cells. These two properties make ESCs remarkably powerful research tools, providing an experimental platform for a) modeling embryonic development in the culture dish and b) transmitting complex genetic modifications through the germ line to make transgenic animals. The development of human ESCs, a potentially unlimited source of specialized cells for studying disease processes in culture and testing new drugs, promises to deliver unparalleled opportunities in regenerative medicine. Notwithstanding their exciting potential, ESCs have only been derived from a limited number of animals. However, the recent development of a novel dual chemical inhibitor (2i) medium that blocks ESC differentiation signals makes it possible to extend ESC technology to other important research animals, such as the rat.
Because of its size, lifespan, physiology, genetics, reproduction and behaviour the rat is one of the most widely used animals for modeling aspects of human behavior and disease. Recent advances facilitating the introduction of targeted mutations using rat ESCs and gene editing enzymes makes it possible to design bespoke genetically modified rat models of human diseases. ESC technology is particularly suited to engineering complex genetic modifications, but can be compromised by instability in cultures. However, recent studies have shown that the ESC state can be stabilised by "epigenetic" modification (not affecting DNA sequence) of genes,. Interestingly, vitamin C, the micronutrient and common food additive, has recently been shown to regulate enzymes that modify the epigenetic status of genes, and stabilize the acquisition of pluripotency. We propose that instability in rat ESC cultures arises from discordance between the undifferentiated state of rat ESC in 2i medium and a more naive "ground state" condition closer to that in the early embryo. In light of Vitamin C's role as epigenetic regulator, we tested its effects on the potency of rat ESCs.
We find that vitamin C increases proliferation of rat ESC, improves their undifferentiated status, and enhances gene targeting. Vitamin C treated cells retain developmental potency as they integrate into host embryos to generate chimaeric animals. Interestingly, the antioxidant D-isoascorbic acid (DiAA), a vitamin C stereoisomer, only stabilises ESC self-renewal, indicating that vitamin C regulates rat ESC by at least two distinct mechanisms.
In this project we will ask how vitamin C affects, 1) gene activity and methylation status of rat ESC, 2) the developmental capacity of rat ESC in vivo and 3) genetic modification of rat ESCs? We will compare gene transcription and methylation profiles of control, vitamin C and DiAA-treated cells to identify vitamin C-regulated genes, and use genetic loss or gain-of-function experiments to test their functional contribution. We will examine the developmental potential of vitamin C treated cultures by analysing their engraftment in adult chimaeras and functional contribution to the germ line. Finally, we will systematically compare gene targeting in different classes of genes, to assess the benefits of Vitamin C in genetic engineering.
The practical aim of these experiments is to improve rat ESC utility. These studies provide insights into the maintenance of pluripotency, and in particular how epigenetic modifications mediated by a widely used food additive affect developmental capacity of ESCs, including germ line differentiation. Cross-species comparisons using the rat ESC data sets will provide novel insights into the evolution of epigenetics and regulation of pluripotency in other mammals.

The rat is one of the most commonly used lab animals in biomedical research, and recent advances in rat embryonic stem cells (ESC) technology present new opportunities for applying state-of-the-art genetic engineering to this model organism. Recent reports describe how vitamin C (ascorbic acid), the recently identified epigenetic regulator, facilitates reprogramming to an ESC-like state. We tested the effect of ascorbic acid on rat ESC and found it stimulated proliferation, reduced differentiation, and appeared to increase gene targeting. The aims of this project are to determine how ascorbic acid affects, 1) gene regulation in rat ESC, 2) developmental potential of rat ESC in vivo, and 3) gene targeting. Our studies have shown that anti-oxidant and enzyme cofactor activities of ascorbic acid differentially affect growth and differentiation of rat ESC. We will compare transcription, and DNA methylation profiles of rat ESC treated +/- ascorbic acid, or a stereoisomer lacking enzymatic cofactor activity, to understand how the vitamin affects gene regulation. We will use functional assays to determine which targets and associated regulatory pathways mediate the ascorbic acid response in rat ESC. Rat ESC contribute efficiently to chimaeras but generally exhibit lower levels of germ line transmission. We will derive new lines +/- ascorbic acid to test how it affects the developmental potential of rat ESC within ESC-derived chimaeras, including transmission through the germ line. We will also compare the efficiency of CRISPR/CAS9-mediated non-homologous end-joining and homology-directed repair at three independent loci, within different classes of ESC genes. In summary this programme will investigate how ascorbic acid improved stability of rat ESC translates into improvements in germ line potential and transgenesis. Our study will also provide important insights into the molecular mechanisms by which this important metabolite affects developmental potential of ESC.

WHO WILL BENEFIT FROM THIS RESEARCH?
The primary beneficiaries of outputs from this research, in the short to medium term will be the scientific community in the academic and industrial sectors. The longer term potential beneficiaries will include the biotechnology and pharmaceutical industry. Products developed by these sectors that are informed or enabled by the knowledge, technologies and resources developed in this research project could ultimately deliver benefits to the wider public. These long-term beneficiaries could include those who would benefit from applying stem cell technology more widely to species in which it still proves challenging to propagate pluripotent stem cells, and those that require treatments for diseases that can be modelled in a physiologically relevant and widely used experimental animal like the rat.

HOW WILL THEY BENEFIT FROM THIS RESEARCH?
The first stage in delivering benefits from this research will be the exploitation of the knowledge and technologies developed in the project. The rat is one of the most common and useful lab animals in biomedical research, and rat embryonic stem cells (ESCs) represent a new opportunity for applying state-of-the-art genetic engineering to this model organism. Vitamin C, the essential micronutrient and recently identified epigenetic regulator, facilitates reprogramming of an ESC-like state. We tested the effect of vitamin C on rat ESC and found it stimulated proliferation, reduced differentiation, and appeared to increase gene targeting. A major goal of this research is to understand better how vitamin C regulates the stability of pluripotent rat embryonic stem cells (ESCs) and thereby maximise the utility of rat ESC in transgenesis. Knowledge garnered from this project will inform researchers on the development of robust methodologies for the propagation and differentiation of both rat ESC. It may also increase the reliability of applying sophisticated genetic engineering techniques to rat ESC, and achieving efficient transmission of genome modifications through the germ line, thereby accelerating the use of rat ESC in transgenesis. The development of more robust protocols for the propagation and genetic manipulation of rat ESC will have direct impact for researchers interested in developing new genetic models in rat, both in academic and industrial contexts.

Improvements in the cultivation and manipulation of rat ESC could also have important implications for propagation of stem cell lines of other species, and therefore be of interest to researchers studying livestock genetics and animal sciences, as well as scientists in the agro-biotechnology industry.
Investigation into effects of vitamin C, and its biologically (functionally) distinct stereoisomer D-isoascorbic acid, both widely used food additives, on embryonic stem cell patterns of gene regulation and developmental potential will inform researchers in academia and the food industry, on potential roles of these additives on the role of epigenetic programming and embryonic potential.


WHAT WILL BE DONE TO ENSURE THAT THEY HAVE THE OPPORTUNITY TO BENEFIT FROM THIS RESEARCH?
In order to ensure that the benefits of this research can be realised we will communicate our results (knowledge and technologies) in a timely manner at scientific meetings and the peer-reviewed scientific literature. We will make genetically engineered rat ES cells available to other research groups under appropriate material transfer agreements and license arrangements. Subject to appropriate funding we will provide training for other researchers in methods developed during the project. We will use the expertise of our technology transfer office and any industrial contacts to seek collaborative project opportunities with industry as well as with academia.