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

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.

The constituent tissues of the eye derive from different primordial cell lineages. The corneal epithelium and lens, for example, are manifestly different tissues, but both derive from surface ectoderm. The heavily pigmented iris and the transparent corneal stroma, on the other hand, have a neural crest origin. Recently, several groups have made progress generating eye tissue -- retina mainly -- via the direct differentiation of induced pluripotent stem (iPS) cells (Reichman et al., PNAS 2014;111:8518-23; Zhong et al., Nat Commun 2014;5:4047). But, there was a step-change recently when our Japan-based collaborators on this proposal discovered that human iPS cells can form cellular multi-zones in culture and that, based on immunostaining patterns, cells in different zones resemble cells of different eye tissues (Hayashi et al., Nature 2016;531:376-80). The finding has fundamental importance, but also significant translational potential, as was illustrated by the fact that a functional iPS cell-derived corneal epithelium recovered vision in an experimentally induced animal model of corneal blindness (Hayashi et al., Nat Protoc 2016; in press). The aim of our research is to provide definitive answers to questions raised by the indicative results of Hayashi et al, working closely with them. Briefly, we will investigate the iPS cell-derived constructs and their cell-cell junctions at high resolution and in 3D, both within cell zones and across zonal boundaries. We will also generate a range of exogenous matrix molecules to simulate the corneal epithelial stem cell niche to enhance iPS cell-derived corneal epithelial expansion, and will probe the translational potential of putative lens-like cells. The research will provide excellent training for a UK-based post-doc, has high value in terms of internationalisation and knowledge transfer, and, we propose, will result in significant advances in our understanding of iPS cell biology, eye development and regenerative medicine.

This proposal has the potential to make a demonstrable contribution to the UK's knowledge economy, to new translational medicine approaches for eye surgery, and to our understanding of human induced pluripotent stem (iPS) cell technology.

Theme I: Public Engagement: The discovery that mature cells can be reprogrammed into iPS cells won the 2012 Nobel Prize for Sir John Gurdon (Cambridge) and Prof Shinya Yamanaka (Kyoto). This has resonance, and we will mention this exciting discovery in our recruitment of students into postgraduate research, highlighting the fact that Prof Yamanaka is collaborating with us. We will engage with the general public to communicate the importance of iPS cell technology at a fundamental and translational level. A symposium in Cardiff in 2020 will feature a Keynote Lecture by Prof Kohji Nishida (Osaka, Japan), our collaborator on this application. The event will be advertised and open to members of the public. Our collaborative research with Prof Nishida and his team leading up to the current application received considerable media interest (selected links are provided below). We were heartened that this was not confined to the scientific press, but ran in the mainstream media, too, with stories in The Daily Telegraph and Wall Street Journal, plus interviews on BBC radio http://bit.ly/AndrewQuantock and local commercial radio. We targeted our public engagement widely and were particularly pleased that several youth-oriented outlets such as Wired Magazine ran with the story. This proactive approach to public engagement will continue.

Theme II: Translational Potential: This research has high translational potential. It is clear that the first move to clinical trials will happen in Japan, but our research jointly with the Osaka and Kyoto teams will significantly inform this move, and we envisage UK take-up after first-in-man trials in Japan. Corneal epithelial regeneration from iPS cell-derived constructs is likely within 18-24 months from the start of clinical trials. The iPS cell-derived lens work is earlier in the translational pathway, but very exciting nonetheless.

Theme III: Commercial Potential: Based on pilot data we hypothesise that variously cleaved motifs of chondroitin sulphate, when coated on culture dishes, will be able to improve the yield of corneal epithelial stem cells in expanded iPS cell-derived corneal epithelial constructs. These constructs are the closest to clinical trials, thus if our hypothesis is correct, there are significant commercial opportunities for us.

Examples of recent public engagement in research underpinning this application.
Daniels JT. Visionary Stem-Cell Therapies. Nature 2016;531:309-310
https://www.sciencedaily.com/releases/2016/06/160630092619.htm
https://www.sciencenews.org/article/new-techniques-regrow-lens-cornea-tissue?mode=magazine&context=188098
http://medicalxpress.com/pdf376743725.pdf
http://www.timeslive.co.za/scitech/2016/03/10/The-eyes-have-it-scientists-grow-lenses-from-stem-cells
http://www.dailymail.co.uk/sciencetech/article-3484134/Have-scientists-cure-BLINDNESS-Tissues-grown-lab-transplanted-eyes-help-restore-sight.html
http://www.walesonline.co.uk/news/health/scientists-found-way-restore-sight-11016715
http://www.telegraph.co.uk/news/health/news/12189238/Scientists-use-stem-cells-to-grow-living-lens-in-eye-and-cure-cataracts.html
http://www.wired.co.uk/news/archive/2016-03/09/stem-cell-eyesight-restore-rabbits.
http://www.gizmag.com/stem-cells-eyeballs-rabbits/42239/
http://gizmodo.com/living-lens-made-from-stem-cells-could-treat-blindness-1763850297
BBC radio interview with Quantock, 9 March 2016. http://bit.ly/AndrewQuantock
http://www.japantimes.co.jp/news/2016/03/10/national/science-health/ips-studies-give-hope-people-vision-loss/
http://www.asahi.com/ajw/articles/AJ201603100071.html
http://www.wsj.com/video/scientists-study-how-stem-cells-could-treat-corneal-blindness/0D19BE2C-411E-4529-BD15-62F49F85C47E.html