The Genomic Basis of Human Induced Pluripotent Stem (iPS) Cell Differentiation into Eye-Like Tissues.

The cells that comprise our body have specific functions and are adapted to suit the particular tissue in which they exist. 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 called differentiated cells because they have become tailored to their biological role in the tissue they help form. 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 (Professor Sir John Gurdon (Cambridge University, UK) and Professor Shinya Yamanaka (Kyoto University, Japan)) for their research, which showed that differentiated adult cells could be genetically reprogrammed to a less differentiated cell, capable of forming many different cell types. Such cells are called induced pluripotent stem cells, commonly abbreviated to iPS cells.

Our new planned research originates from a discovery made by our long-term collaborators in Osaka University, Japan, Professors Kohji Nishida and Ryuhei Hayashi, working with Professor Yamanaka in nearby Kyoto University. Their work showed that human iPS cells can grow in the laboratory to form a disc in which cells in different areas resemble cells found in different parts of the eye, such as the lens, retina and cornea. This discovery is exciting because it will allow scientists to conduct sophisticated experiments to better understand human eye development. The eye-like cells obtained from the human iPS cells also have the potential, in the future, to be used in new treatments for a large variety of eye disorders. Excitingly, the Osaka team showed that iPS-derived cells that most closely resemble the outer surface of the eye known as the corneal epithelium, were able to restore vision in a model of corneal blindness. So, eye-like tissues can be grown in the laboratory from human iPS cells, but we need to comprehensively understand what genetic processes drive this because currently these are unknown.

We will conduct research to determine the complete repertoire of eye-like cell types (e,g. cornea, lens or retina) that can be produced from human iPS cells using the methods discovered by our collaborators, Professors Nishida and Hayashi. This will represent a major new collaboration that will use the latest technologies to understand the genetic drivers of human iPS cell growth and differentiation into eye-like tissues. We will also use the human iPS cells to study an important gene, called TCF4, which is damaged in the most common corneal blinding disease in the world, called Fuchs' endothelial corneal dystrophy. Crucially, the new information will help us understand the mechanisms by which healthy corneal endothelial cells function and what happens when they malfunction, knowledge that will lay the ground for future studies of novel human iPS cell-based therapies. Overall, this collaboration with our colleagues in Japan will lead to significant advances in our knowledge of human eye development, corneal cell biology and the genetic basis that underpins human iPS cell differentiation.

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 pigmented iris and transparent corneal stroma, on the other hand, have a neural crest origin. Recently, our collaborators on this proposal discovered that human iPS cells can form cellular multi-zones in culture, and that cells in different zones resemble cells of different ocular tissues; lens, retina, cornea, etc. (Hayashi et al., Nature 2016;531:376-380). This finding has fundamental importance to help us more fully comprehend human eye development, but also has significant translational potential as was illustrated by the fact that a functional iPS cell-derived corneal epithelium was able to recover vision in an experimentally induced animal model of corneal blindness (Hayashi et al., Nat Protoc 2017;12:683-696). At present, our appreciation of human iPS cell-derived eye-like multi-zones is limited to how the cells look and behave and what molecular markers they express based on immunostaining patterns. We know virtually nothing about the genomic influences that underpin the differentiation of the iPS cells into a diverse range of eye-like tissues. The research proposed here seeks to redress this. We will work closely with our colleagues in Japan to ascertain the genomic drivers and developmental trajectories that guide the formation of human iPS cell-derived eye-like multi-zones. This will be achieved using single cell RNA-sequencing and open-chromatin profiling. We will also identify the transcriptional drivers of cell identity in human iPS cell-derived eye-like multi-zones at single cell resolution, and conduct experiments to establish the role of the gene TCF4 as a master regulator of corneal endothelial cell proliferation and function. The research will lead to significant advances in our knowledge of human iPS cell behaviour and human eye development.

Theme I: Knowledge and People Transfer. Our planned research will make a demonstrable contribution to the UK's knowledge economy based on the high-level transferable skill set in human iPS cell technology that will be acquired by the PDRA in world-leading laboratories in Osaka and Kyoto.

Theme II: Public Engagement. The discovery that mature cells can be reprogrammed into iPS cells, which won the 2012 Nobel Prize for our technical advisor on this project, Professor Shinya Yamanaka, has resonance within the scientific community and beyond, and we will engage with the general public to communicate the importance of iPS cell science at a fundamental and translational level. Our collaborative research with Professor Kohji Nishida, and his team leading up to the current application received considerable media interest (selected links are provided below as examples of where this was communicated), and 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 and 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. Likewise, the sold-out "Eye for an Eye" Pint of Science event in Bristol in 2017 (https://pintofscience.co.uk/event/an-eye-for-an-eye) at which the PI debated this research was highly successful, confirming the public's interest in eye research and stem cell science. Our proactive approach to public engagement will continue and we will communicate and debate our new human iPS cell genomic research via established mechanisms at the MRC Centre for Neuropsychiatric Genetics and Genomics at Cardiff University, which has a well-developed programme of public engagement. We will also empower our PDRA to target Wellcome Trust ISSF public engagement funding (£5K), which will provide him/her with valuable training in this important aspect of contemporary academia.

Theme III: Translational Potential: This research has high translational potential and first-in-man grafts of human iPS cell-derived corneal epithelial constructs will be conducted by our collaborators in Osaka University in the spring of 2019 with the approval of the Japanese Ministry of Health & Welfare. Via our close relationship with our colleagues in Osaka, we will be very well placed to inform relevant UK clinical and regulatory bodies to help map out a pathway for future implementation of human iPS cell-based corneal epithelial surgery in the UK. The iPS cell-derived corneal endothelial/TCF4 work is earlier in the translational pathway, but developing human iPS-derived endothelial constructs for surgical use has significant promise for the future treatment of vision loss due to corneal endothelial dysfunction.

Theme IV: Commercial Potential: IP for the development of human iPS cell-derived corneal epithelial constructs is held by our co-applicants in Osaka University. Shared IP will be investigated moving forward, based on the role of TCF4 in the generation of corneal endothelial cell constructs from human iPS cells, and the future use of human iPS cell-derived corneal stromal cells in the field of regenerative medicine and corneal tissue engineering.

Selected Links as Examples of Dissemination
http://www.timeslive.co.za/scitech/2016/03/10/The-eyes-have-it-scientists-grow-lenses-from-stem-cells
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.
BBC radio interview with Quantock, 9 March 2016. http://bit.ly/AndrewQuantock
http://www.wsj.com/video/scientists-study-how-stem-cells-could-treat-corneal-blindness/0D19BE2C-411E-4529-BD15-62F49F85C47E.html