Anti‐apoptotic properties of mesenchymal stem cells in mouse model of corneal injury

Abstract

AbstractLimbal stem cell deficiency (LSCD) is associated with the break of immune privilege of the cornea, a harmful local inflammatory reaction and a loss of corneal epithelium cells caused not only by damage after injury but also by the apoptosis in later phase. Apoptosis is caused by various factors including damaging of cell structures and chronic inflammation and may be one of the main causes of the failure of corneal regenerative mechanisms. Mesenchymal stem cells (MSCs) turned out to be a suitable source of stem cells for the therapy of bilateral LSCD. MSCs are well known for their anti‐inflammatory, anti‐apoptotic, regenerative, migration and differentiation properties. Antiapoptotic mechanisms are still not well described but their recognition could help for a long‐term regeneration of corneal epithelium after the injury or after the corneal transplantation in cases like LSCD.MSCs were obtained from murine bone marrow. Flow cytometry was used to characterize phenotypic markers of purified MSCs. For in vitro experiments, MSCs were cultured in plates for 24 hours. Excised corneas were added to the culture alone or in inserts and were stimulated by pro inflammatory cytokines. After a 48‐hour co‐cultivation the expression of genes for pro apoptotic and anti‐apoptotic molecules in corneas was detected using qPCR. Flow cytometry was used to detect phosphatidylserine on the surface of apoptotic cells by annexin V. In in vivo experiments, MSCs were seeded onto nanofiber scaffolds (allowed to adhere for 24 hours) and transplanted on the injured cornea (alkali burn)MSCs significantly decreased the number of apoptotic cells in the cornea with simulated inflammation in vitro or in injured corneas in vivo. MSCs also supressed the expression of the pro‐apoptotic molecules Bax and p53 and change the ratio of Bcl‐2 Bax expression in the cornea.Autologous MSCs suppressed apoptosis in in vitro and in vivo models of corneal injury and inflammation.