Michael O'Connor

Lens Research Laboratory, Department of Anatomy, University of Sydney Summary of the Awarded Paper

For some time after the completion of my Undergraduate studies I was intrigued by the idea of replacing fatigued or damaged organs without the need for organ donation. However, it wasn't until I came across an opportunity at the University of Sydney, Australia, that I was able to undertake a tissue engineering project, in a laboratory studying the ocular lens. Members of this laboratory, headed by Prof. John McAvoy, are expert at the technique of culturing explanted lens epithelial cells, having used it to show that fibroblast growth factors (FGFs) could stimulate lens epithelial cells to differentiate into lens fibres. As the laboratory was interested in investigating the role of FGFs in the development of overall lens structure, I enrolled as a PhD student on a project aimed at reconstructing a lens in vitro.

The lens is a bi-concave tissue, encapsulated by a basement membrane, and composed of an anterior monolayer of epithelial cells and a mass of tightly packed fibre cells. The lens fibres develop through the differentiation of the epithelial cells located at the lens equator. During this process, the lens develops the unique structural features that enable it to function. For instance, the developing fibre cells dramatically increase in length, becoming closely packed with one-another to form the highly regular cellular arrangement found in the bulk of the lens. At the same time, the fibre cells express large amounts of specialized cytoplasmic proteins, such as a, ß and y crystallins. Together, the close packing of elongated fibre cells along with the expression of the crystallins, provide the lens with the refractive index required for focusing. Importantly, the developing fibre cells degrade their organelles as they mature, including the nucleus, ensuring that these potentially light-scattering particles are unable to disrupt the transmitted light as it passes through to the retina.

These developmental and structural features of the normal lens were used as markers to assess a number of novel lens epithelial explant culture systems that were developed during the course of my project. The first of these culture systems is capable of generating a transparent, concave tissue containing an anterior epithelial monolayer overlying a small mass of elongated, parallel-aligned fibre cells. An intriguing aspect of this work was the demonstration that only explants cultured with the ocular fluid, vitreous, developed into transparent tissues. When used under the same conditions, explants cultured with FGFs alone developed elongated, parallel-aligned fibre-like cells, however these cells degenerated to leave an opaque tissue with few epithelial cells.

Another culture system that was developed arose from the desire to generate the entire lens structure in vitro, and was based on reproducing aspects of embryonic lens development in culture. During embryogenesis, neuroectoderm is stimulated to thicken and invaginate, creating a spherical monolayer of epithelial cells (the lens vesicle) from which the lens develops. The first lens fibres appear within this lens vesicle when the cells closest to the optic cup are stimulated to differentiate by factors present in the vitreous. Since the lens vesicle is composed solely of epithelial cells and represents the first stage at which lens tissue becomes isolated from other tissues, it was targeted as the starting point for lens reproduction in culture. Because the explant culture system is based on explanted epithelial cell monolayers, it was thought that a lens vesicle might be approximated by placing one explant directly on top of another, with the cells of the two explants in contact with each other. Vitreous was then used to stimulate differentiation within these in vitro lens vesicles. In this way, we were able to generate physiologically-sized, transparent tissues which focus light and magnify images. These functional tissues contain structural, ultrastructural and molecular features similar to those seen in the normal lens. For example, they contain both epithelial- and fibre-like cells, with the differentiating fibre cells being long, thin and parallel-aligned. These differentiating cells also develop intricate membrane structures and show evidence of terminal denucleation, typical of lens fibres in vivo. However, after approximately 45 days of culture, these tissues develop an opacity which is biomicroscopically and ultrastructurally similar to age-related human cataract. Since the progression of molecular changes responsible for age-related cataract are poorly understood, this culture system could become a valuable tool in understanding human cataract development.

Both the transparent and focusing culture systems use vitreous fluid to supply growth factors. Although FGFs are known to be present in the vitreous and are able to stimulate differentiation within explants, they were unable to produce transparent or focusing tissue when used in these culture systems. To determine what other factors are required to generate functional lens tissue in vitro, a proteomic analysis of lens membrane proteins is being undertaken. We are interested in seeing whether it is possible to identify growth factors involved in lens development by identifying the growth factor receptors present in lens cells. The long-term aim of this project is to be able to replace the vitreous fluid used to generate the in vitro lenses with a defined set of growth factors at specific concentrations. Should this be possible, it would then open up the exciting prospect of systematically analysing the molecular steps involved during lens development and cataract formation.

At present, there are estimated to be 38 million people throughout the world who are blind due to cataract. Although surgery, which is currently the only effective treatment for cataract, can restore sight to the patient, it often results in secondary cataracts that require further treatment. Unfortunately, surgical treatment for both cataract and secondary cataract are limited in less developed countries of the world. This, in combination with the increasing average age of the worlds population, have led the World Health Organization to estimate that the number of people suffering from cataract will dramatically increase over the coming decades. Consequently the need to find non-surgical ways of managing cataract is of major importance. In this context, the functional lenses we have developed in culture may prove beneficial, by helping to reveal the molecular details responsible for lens development.