Stem cell organisation and differentiation in the niche

There is an expectation that stem cells will one day be able to cure a wide variety of human diseases. However, development of stem cell therapies relies upon a sound understanding of the biology of these fascinating cells. Stem cells occupy a specialised environment in the body known as a ?niche? which controls stem cell behaviour and survival. Most human stem cell niches are deep in the body and so difficult to study. The outermost layer of the cornea on the front surface of the eye has stem cells which live in a transparent area called the limbus. We have recently discovered new structures in the limbus which we believe may be the stem cell niche. We propose to study these structures and the cells associated them in donated human corneas using techniques including scanning electron and confocal microscopy determine where exactly the stem cells are and how they function. We will also study the effect of age upon the stem cells and their niche. This is very important because clinically it is possible to take a small biopsy of the healthy limbus to prepare stem cell therapy for patients blinded by specific injuries or diseases of the outermost layer of the cornea. However, we and others are providing this therapy without knowing the optimal biopsy location. Our study will therefore improve this surgical technique. We must also be able to refine treatments for individual patients which requires knowledge of what happens to the stem cells after they have been transplanted. We propose to develop a technique for labelling and transplanting stem cells onto the human cornea using very small particles which fluoresce brightly and can be detected with clinical instruments. Using this methodology we will also determine how well stem cells organise themselves in the niche after transplantation and if they have any affect upon that niche. Our data will help us to understand one of the important features of the eye which contributes to clear vision (i.e. healthy, functioning stem cells in the limbus) which will help to improve stem cell therapy for the cornea. If it is found that our data provide parallels with other human stem cell niches, it would support the use of the readily accessible and transparent cornea as a model for the development of other stem cell therapies.

This research proposal reflects the current scientific and clinical focus on stem cells in health and disease. Occupation of a specialised niche environment is important for stem cell survival and function; however, in the main these niches are difficult to study in humans. In the transparent cornea, a population of limbal epithelial stem cells (LESCs) constantly regenerates the epithelium providing an excellent model for studying an adult stem cell niche. LESC deficiency is a serious blinding condition with no accepted cure. Cultured LESC therapy is being trialled clinically in laboratories around the world including ours. However, the characteristics of this stem population and its niche (the limbus) are poorly understood. Two of the key elements of LESC therapy efficacy are the quality of the biopsy from which LESC are originally harvested for in vitro expansion and the condition of the recipient host environment, both of which are have been hampered by a lack of technology to clearly visualise the niche and transplanted cells over time. We have recently identified novel structures (crypts and focal stromal projections) within the limbus which we propose to be a putative LESC niche owing to the characteristics of the resident cells. We propose to build upon these findings to further test our hypothesis of the existence of heterogeneity in the LESC niche and also determine the effect of age upon the cellular and architectural components. These studies have particular clinical relevance as they will provide data to direct the location from which a limbal biopsy should ideally be taken for LESC therapy production. Currently biopsy harvest location is not based on experimental evidence. Furthermore we will evaluate the practicality of using fluorescent semi-conductor nanocrystals (QDots) for labelling and tracking of transplanted cells on the human cornea using an ex-vivo culture system ? a technology with future clinical potential. Specifically we will assess the ability of the de-cellularised LESC niche to support the re-organisation of cultured limbal epithelial cells, including LESC, to their locations in normal tissue. The ?potency? of the niche for directing stem cell fate will be tested using GFP labelled mouse embryonic stem cells. Finally, we will assess the regenerative capacity of the LESC niche in our model systems. This is an ambitious element of our work but one which may shed light on the mechanisms of LESC therapy efficacy and failure which are currently not understood.