The cornea is the transparent outermost surface of the eye, consisting of a stratified epithelium, a collagenous stroma and an innermost single-cell layered endothelium and providing 2/3 of the refractive power of the eye. Multiple diseases of the cornea arise from genetic defects where the ultimate phenotype can be influenced by cross talk between the cell types and the extracellular matrix. Cell culture modeling of diseases can benefit from cornea organoids that include multiple corneal cell types and extracellular matrices. Here we present human iPS cell-derived organoids through sequential rounds of differentiation programs. These organoids share features of the developing cornea, harboring three distinct cell types with expression of key epithelial, stromal and endothelial cell markers. Cornea organoid cultures provide a powerful 3D model system for investigating corneal developmental processes and their disruptions in diseased conditions.

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Corneal epithelium is maintained throughout life by well-orchestrated proliferation of limbal epithelial stem cells, followed by migration and maturation centripetally across the ocular surface. The present study sets out to explore the role tissue stiffness (compliance) may have in directing both differentiation and centripetal migration of limbal epithelial stem cells during homeostasis. For that, we analysed the localization of the Yes-associated protein (Yap), a transcriptional co-activator previously shown to mediate cellular response and mechanical stimuli. Using both models of ocular surface compliance and normal bovine corneas we evaluated the nuclear/cytoplasmic expression ratio of Yap. Expression levels within corneal epithelial cells were compared in situ between the limbus and central cornea, and in vitro between limbal epithelial stem cells expanded upon biomimetic collagen gels of increasing stiffness. Nuclear expression of Yap was shown to increase within the expanded cells upon substrates of increasing stiffness. Subsequently, Yap was used as a novel molecular probe to investigate the mechanical microenvironment within a normal ocular surface. The in situ localization of Yap was predominantly cytoplasmic within basal limbal epithelial cells and nuclear within basal central corneal epithelial cells. Furthermore, nuclear p63 expression was not co-localized with Yap in basal limbal epithelial cells. In conclusion, the current investigation provides new insights into the relationship between Yap and distinct cell populations across the ocular surface indicating that cells experience a different mechanical environment between the limbus and central cornea. A new hypothesis is put forward, in which centripetal differences in substrate stiffness drives the migration and differentiation of limbal epithelial stem cells, thus controlling corneal epithelium homeostasis.

The avascular cornea is a uniquely-isolated organ, with its stroma constituting a nutrient-poor environment. Consequently, the availability of metabolites such as glucose to corneal stromal cells is considerably reduced compared with other tissues, or indeed with media commonly used to culture these cells in vitro. However, the role of glucose in the behaviour of human corneal keratocytes has been overlooked. As such, we sought to investigate the effects of low-glucose formulations on the phenotype of human corneal stromal cells. Cells cultured in low-glucose were able to survive for extended periods when compared to high-glucose, serum-free conditions. Furthermore, low-glucose enhanced their reversal to a keratocyte-characteristic phenotype. Specifically, cells within low-glucose medium assumed dendritic morphologies, with bean-shaped condensed nuclei, absence of alpha-smooth muscle actin or stress fibres, and a corresponding reduction in migratory and contractile activities when compared with high-glucose, serum-free conditions. Moreover, cells within low-glucose uniquely recovered the ability to express a robust keratocyte-characteristic marker, CD34, while still expressing elevated levels of other representative phenotypic markers such as keratocan, lumican, ALDH1A1, and ALDH3A1. These results indicate that low-glucose enhances keratocyte-characteristic phenotype above and beyond established media formulations and thus has important implications for corneal biology in health and disease.

Keratoconus is a common corneal ectasia that leads to progressive visual impairment. Numerous studies have shown abnormal protein expression patterns in keratoconic corneas. However, the specific mechanisms causing this disease remain ambiguous. This review aims to provide an update on morphological studies of the keratoconic cornea, relate these early studies with current findings from proteomic, biochemical and cell culture studies and to postulate possible pathogenic pathways.