The Development of Eye Tissues via Human Induced Pluripotent Stem (iPS) Cells.

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The cells that exist in the tissues of our body have specific functions and are adapted to suit the particular tissue that they help form. Skin cells, for example, are different to eye cells, which are different to blood cells. And, of course, each cell type has its own distinctive role. Mature cells, whichever tissue they are in, are what we know as differentiated cells, because they have become adapted to their biological role in the tissue in which they help form. But, during development in the embryo our cells do not have this tissue-specific identity. Indeed, the early cells in embryogenesis can go down different developmental pathways and become different cell types. These early undifferentiated cells are known as stem cells.For a long time, it was accepted that once a cell had "chosen its path" and differentiated into a particular type of cell, it had embarked on an irreversible process. But, in 2012 two scientists were awarded the Nobel Prize in Physiology or Medicine (Sir John Gurdon (Cambridge University, UK) and Prof Shinya Yamanaka (Kyoto University, Japan)) for their research, which showed that differentiated adult cells could be reprogrammed via a manipulation using four genetic factors added to a cell's nucleus - factors that are now known as Yamanaka factors. These reprogrammed cells became an earlier, less differentiated cell, capable of differentiation into a number of cell types. Such cells are called iPS cells -- induced pluripotent stem cells.Our new planned research is based on a discovery made by our collaborators on this proposal in Osaka University, Japan, working with us and with Prof Yamanaka and his team in nearby Kyoto University. It showed that some types of human iPS cells can grow in the laboratory and form a cellular multi-zone, in which cells in different areas resemble cells of different eye tissues; lens, retina, cornea, for example. This discovery is particularly exciting because the cells that most closely resemble a natural corneal epithelium - i.e. the front layer of cells on the eye that support the tear film - can be transplanted onto the eye where they remain functional. This research has a strong future potential for the use of human iPS cells for the treatment of eye disease.In our planned study we will investigate the iPS cells that have formed into eye-like tissue. To do so, we will use powerful 3D electron microscopy technologies that can image the cells at high resolution and at high magnification. We will also use antibodies to specific molecules to see how the iPS cell-derived eye-like cells are communicating with one another, and how we can manipulate these communication pathways using certain chemicals to modulate the formation of eye-like iPS cells. We also have expertise from our previous experiments on cornea and cartilage in understanding how some molecules, called proteoglycans, help keep stem cells to stay stem cells in their niche areas or environments in tissues. In the healthy cornea, for example, we know that various special types of proteoglycans populate an area of the cornea known as the stem cell niche. Basically, this is a region of a tissue that helps support and maintain the stem cells. We will now extract and purify proteoglycans from cornea and chemically modify them using enzymes. The modified proteoglycans will then be used to help grow iPS cell-derived corneal epithelial constructs to understand what types of proteoglycans or cleaved proteoglycan fragments are important to retain cellular "stemness". This will help improve the potential for human iPS cell-derived corneal epithelial cell production for future surgical use. Finally, and importantly, we will examine in detail how iPS cells in our eye-like multi-zone can be used to replicate lens cells, which has the exciting potential of helping the regeneration of lenses in the human eye.