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

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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.