Investigation of the interaction of limbal epithelial cells and stromal cells of patients with aniridia-associated keratopathy

• Applicant: Dr. Tanja Stachon
• Subject Area: Ophthalmology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 522478626

Congenital aniridia is a rare disease of the eye, affecting approximately 1 in 64000 of the population. Patients who have aniridia due to a mutation of the highly conserved PAX6 gene, which is relevant for ocular development, suffer from an early onset of aniridia-associated keratopathy (AAK), among other symptoms. This leads to massive corneal problems (epithelialisation disorders, recurrent erosions, vascularised corneas). The possible causes of AAK include stem cell loss, stem cell dysfunction or impaired interaction between keratocytes (the stromal cells of the cornea) conjunctiva and epithelial cells. For previous studies, we have isolated and cultured limbal epithelial cells from the limbal region of aniridia patients with advanced AAK. Similarly, it is possible to isolate and culture the limbal stromal cells. Little is known about these cells so far, and in our studies we would like to find out more about the interaction of the epithelial and stromal cells of the limbal region. In addition, we would like to investigate how the epithelial and stromal cells react to inflammatory stimuli and oxidative stress in cell culture. To gain more insight into the genetic expression pattern of the epithelial and stromal cells of aniridia patients, microarray analysis is an important tool to generate a systematic basis for further investigations. As the possibility to use biopsy material from patients with aniridia for experimental work is limited due to the very rare disease pattern, we depend on working with cell models. In our research group, we have developed a PAX6-siRNA aniridia-based epithelial cell model. We would like to use this cell model (it mimics the epithelial cells of aniridia patients) if it becomes foreseeable that not enough cells from aniridia patients are available. A single cell suspension is produced enzymatically from tissue biopsies. Here, the epithelial cells can be separated from the stromal cells and subsequently cultured. If there are not enough biopsies from aniridia patients, biopsies from deceased patients who have provided their corneas for transplantation are used and from whose cells we produce a si-RNA-PAX6 cell model. After induction of inflammatory stimuli and hypoxic stress, or co-cultivation of limbal epithelial and stromal cells, inflammation-relevant markers as well as stem cell and differentiation markers are measured and evaluated from the cultured cells using mircroarray, qPCR, WB and/or ELISA.

• DFG Programme: Research Grants