Ex vivo 3D human corneal stroma model for Schnyder corneal dystrophy - role of autophagy in its pathogenesis and resolution
Dóra Júlia Szabó1, Richárd Nagymihály1, Zoltán Veréb1, Natasha Josifovska1, Agate Noer2, Petra Liskova3, Andrea Facskó1, Morten C. Moe2 and Goran Petrovski1,2
1Stem Cells and Eye Research Laboratory, Department of Ophthalmology, Faculty of Medicine, University of Szeged, Szeged, Hungary, 2Center of Eye Research, Department of Ophthalmology, Oslo University Hospital, University of Oslo, Oslo, Norway and 3Insitute of Inherited Metabolic Diseases, First Faculty of Medicine, Charles University and General University Hospital in Prague, Czech Republic
Offprint requests to: Goran Petrovski, MD, PhD, Dr habil, Korányi fasor 10-11, 6720, Szeged, Hungary. e-mail: email@example.com
Summary. Introduction: Multilamellar bodies (MLBs) are concentric cytoplasmic membranes which form through an autophagy-dependent mechanism. In the cornea, the presence of MLBs is associated with Schnyder corneal dystrophy (SCD). Ex vivo 3D modelling of the corneal stroma and SCD can help study pathogenesis and resolution of the disorder.
Methods: Corneal stroma explants were isolated from cadavers and cultivated long-term for more than 3 months to achieve spontaneous 3D outgrowth of corneal stroma-derived mesenchymal stem-like cells (CSMSCs). The 3D tissues were then examined by transmission electron microscopy (TEM) for presence of MLBs, and by immunofluorescent labelling against markers for autophagy (p62, LC3). Autophagy was induced by classical serum starvation or rapamycin (RAP) treatment (50nM), and inhibited by the autophagy inhibitor 3-methyladenine (3-MA, 10mM) for 24 hours.
Results: CSMSCs can form spontaneously 3D outgrowths over a 3-4 weeks period, depositing their own extracellular matrix containing collagen I. TEM confirmed the presence of MLBs in the long-term (>3 months) 3D cultures, which became more abundant under starvation and RAP treatment, and decreased in number under autophagy inhibition with 3-MA. The presence of autophagy and its disappearance could be confirmed by an inversely related increase and decrease in the expression of LC3 and p62, respectively.
Conclusions: MLB formation in long-standing CSMSC cultures could serve as a potential ex vivo model for studying corneal stroma diseases, including SCD. Inhibition of autophagy can decrease the formation of MLBs, which may lead to a novel treatment of the disease in the future. Histol Histopathol
Key words: Corneal stroma-derived mesenchymal stem-like cells (CSMSC), Schnyder corneal dystrophy, 3D model, Autophagy