Role of nitric oxide in the maintenance and differentiation of human embryonic stem cells

One of the unsolved fundamental questions in biology is how a single cell, the fertilised egg develops into all the different types of cell in the adult body. Stem cells are central to this process. Human embryonic stem (hES) cells are derived from the blastocyst, a hollow ball of cells formed about a week after fertilisation in the human. hES cells have the potential to turn into all cells of the body and present a potentially unlimited source of cells to treat degenerative diseases such as Alzheimer?s disease, Parkinson?s disease, multiple sclerosis and type 1 insulin-dependent diabetes. However, before hES cells can be used to treat human diseases, we need to understand how to maintain these cells efficiently in culture as well as to control their specialisation into clinically useful cell types, such as insulin-producing pancreas cells in the case of patients with diabetes. Nitric oxide is an important signalling molecule involved in the regulation of cellular function. This proposal aims to understand the role of nitric oxide in hES cells. It is proposed that nitric oxide is a critical factor in the maintenance of hES cells and increased levels control differentiation to specialised cell types. Ultimately, this research will provide important information on how to direct hES cells into clinically useful cells and enhance the provision of new therapies for patients suffering from currently incurable diseases.

Human embryonic stem (hES) cells are pluripotent cells derived from the inner cell mass of the blastocyst. They proliferate by a process of self-renewal and have the potential to differentiate into all cells of the body. Thus, hES cells may offer an unlimited source of cells for transplantation to cure patients with degenerative diseases such as Alzheimer?s disease, Parkinson?s disease, multiple sclerosis and type 1 insulin-dependent diabetes. However, prior to their clinical use, we must first increase our knowledge regarding the maintenance of hES cells and how their differentiation may be directed down specific lineage pathways. Research in this proposal aims to investigate whether the level of NO production is a critical factor influencing hES cell metabolism and phenotype. This will be achieved by determining the importance of NO in the maintenance of pluripotency, whether increased levels direct differentiation down a specific lineage pathway, particularly into cardiomyocytes and whether NO has a role in regulating hES cell metabolism. The effect of modulating NO production on hES cell differentiation will be determined with the use of NO donors, NOS inhibitors, NO scavengers and RNA interference. In addition, the ability of NO to compete with oxygen for binding to cytochrome c oxidase and up-regulate glycolysis will be determined to investigate the effect of NO in the control of enegy metabolism in pluripotent and differentiated cells. This project will use a variety of techniques including laser scanning confocal microscopy, FACS analysis, molecular and protein biology as well as biochemical assays. The hypothesis is that NO is a critical regulator in the maintenance of hES cells and high levels direct differentiation towards a cardiac lineage. Research in this proposal will characterise mechanisms involved in hES cell maintenance and differentiation.