Understanding the molecular and cellular complexity of human cornea through single cell analyses

The cornea is the clear window at the front of the eye that allows light to enter and be focused on the back of the eye. To see a clear image, the cornea needs to be transparent, regular and smooth. Presence of cataracts, corneal damage and corneal diseases are the largest cause of corneal blindness which accounts for 23 million people worldwide adding a huge burden to patients and health care resources. Often the only treatment option is surgical transplantation of donor cornea, a therapeutic option that has been unchanged for more than 50 years. In Europe, over 40,000 blind people are waiting for corneal transplant every year. This shortage results in about 10 million untreated patients globally and 1.5 million new cases of blindness annually.

The outer layer of the cornea (the epithelium) is continuously replaced due to normal cell shedding in response to blinking and physical and environmental damage. Limbal stem cells (LSCs) endlessly produce new epithelial cells in the cornea and prevent conjunctival epithelium (a covering of the white part of the eye) from migrating over the cornea. If these LSC are damaged or diseased, a condition called limbal stem cell deficiency (LSCD) occurs, causing discomfort and reduced vision. In patients with LSCD in one eye, LSCs can be removed from their other healthy eye and grown in a laboratory and then transplanted back into the diseased eye to restore the stem cells and their vision. Using this technique, we have been able to successfully treat 33 patients with LSCD during the last 14 years. Despite this success, we do not know yet how to identify and purify individual LSCs. Moreover, the growth of LSCs under laboratory conditions is significantly limited and they rapidly lose their ability to grow continuously. For this reason, cells used for transplantation are a mixture of LSCs and other cell types which can vary from patient to patient and from one research group to another depending on the technique used for their expansion. The number of LSCs is crucially important for the success of clinical transplantation; for example patients transplanted with laboratory expanded cultures which contain less than 3% of LSCs had a successful transplantation in only 11% of the patients, while those with more than 3% had a successful transplant in 76% of the patients. It is essential to assess the fraction of LSCs before transplantation so unsuccessful transplants can be avoided. To achieve this, we need to know more about LSCs themselves.

Stem cells have also been found in other parts of the cornea, for example the middle part called stroma, and the endothelium which keeps the cornea hydrated. We don't know if stem cells in each of these layers behave the same way as each other or whether there are several types which respond in different ways to corneal damage. Great advances in technologies that allow single cells to be studied individually have enabled development of the Human Cell Atlas, which is currently focusing on tissues such as skin and blood. In this project, we propose to focus on three different regions of adult cadaveric human cornea (central, peripheral and limbal) which have been shown to differ in cell composition and density. The single cell analysis will enable us to know which genes are expressed in every cell and why and to understand how many cell types are in every region and every layer. Since our eyes develop mostly before we are born, we also propose to look at samples from aborted specimens which are donated for research with mother's consent. By understanding the complexity of cell types from development to adulthood, we will better understand how cornea is formed, how stem cells maintain corneal homeostasis and how we can increase the success of the clinical transplantations. The ultimate impact of this project will be to contribute towards safer and more efficacious treatments of a significant proportion of world blindness caused by corneal damage and disease.

Cornea is the transparent front part of the eye which together with the lens focuses the light onto retina for visual processing. Corneal blindness is the 2nd main cause of blindness worldwide accounting for 23 million patients. In Europe, over 40,000 blind people are waiting for corneal transplant every year. Despite efforts to develop corneal substitutes to alleviate both the shortage and the drawbacks of human donor tissues, surgery with allogeneic epithelial donor tissue has remained the gold standard; however this does not work for corneal diseases caused by the dysfunction of stroma or endothelium. Developments in bioengineered corneal substitutes designed to replace diseased corneas have been reported; however their clinical use has been limited due to multiple challenges including lack of integration into the surrounding tissue, complex implantation procedures and serious complications. Clearly, there is an unmet need for the design of new smart biomaterials and stem cell therapies to create corneal structures that are indistinguishable from the original native tissue and fulfill the natural functions of the cornea. By understanding the physical and cellular structure of the tissue and the resulting impact of the structure on biological function, novel materials for a number of ophthalmic clinical applications can be designed. Furthermore, by extending this structure-function approach to characterizing corneal disease processes, new therapies can be engineered. This application aims to utilize the most recent advances in single cell sequencing to reveal the intricacies of corneal structure at the cellular and molecular level from early development to adulthood. Data generated from this project will be deposited into Data Coordination Platform contributing to the Human Cell Atlas and providing the scientific and clinical community with comprehensive datasets that can be further mined and exploited in the context of normal development and corneal disease.

The completed research will have outputs that will have the following impacts, benefiting both basic and translational research:

1) Data sharing
The research programme will generate large amounts of RNA-Seq and ATAC-Seq data which will be deposited into the Human Cell Atlas (HCA) Data Coordination Platform (DCP) and European Nucleotide Archive and analysed following established pipelines. The HCA- DCP will deposit data into community approved archives, under the HCA creative commons by attribution license, making the data immediately accessible and reusable by other research groups.

2) Basic Biology
The research programme will generate significant amount of data on human corneal development, pinpointing stem cell emergence and subsets as well as their gene expression and epigenetic signatures. These will be extremely valuable for our understanding of human development and identification of master genes that upon dysregulation lead to human corneal disease.

3) Publications
We will disseminate our research findings and data-sets to the scientific community through presentations at national and international conferences. All published work will be open-access and will be deposited in PubMed Central Universities' ePrints facilities, Research Gate, etc. in accordance with RC-UK, HEFCE and EU policies. Publications will acknowledge the support received from MRC-UK.

4) Clinical and translational research advances
Corneal disease and damage have been estimated to cause blindness in 8 million people worldwide each year and the cost of this may be as much as £150 billion per year. In cases of bilateral LSCD, most patients are registered as severely sight impaired which requires social support for visual loss, making this disease both costly and affecting the patient's quality of life. The World Health Organisation estimates that one year with severe visual impairment is equivalent to the loss of 23 weeks of life in perfect health. The immediate impact of our project lies in detailed molecular and cellular phenotyping of all corneal cell layers using the most advanced single cell sequencing technologies from development to adulthood. This will provide an important leap in our understanding of stem and progenitor cells that maintain corneal homeostasis, their emergence and differentiation. Furthermore, it will lead to identification of cell markers that can be used for their selection prior to transplantation and future quantitative and qualitative assessments of the stem cell content of grafts before transplantation. Understanding of the corneal structure in such finite details will enhance tissue engineering efforts to generate corneal structures that are indistinguishable from the original native tissue and fulfill the natural functions of the cornea. Its ultimate impact will be to contribute towards safer and more efficacious treatments of a significant proportion of world blindness caused by corneal disease.