Abstract: The meibomian gland is a holocrine gland that secrete lipid meibum and protect ocular surface from desiccation. Meibocytes are lipid synthesizing cells, their maturation process involves lipid accumulation and cell disintegration to release cellular content as meibum. To maintain lifelong meibum production, continuous meibocyte regeneration is required but the understanding of the meibocyte replenishment from meibocyte stem/progenitor cell is limited. We studied two Iroquois mouse mutants (Irx3 and Irx5) that exhibit meibomian gland atrophy and dry eye disease features. We successfully established a mouse meibomian gland organoid culture that displayed meibocyte differentiation, the organoids could be expanded for over 20 passages. Using this meibomian gland organoid culture platform, we investigated the roles of Irx3/5 genes in meibocyte differentiation and maturation. Mouse meibomian glands were isolated and cultured in three-dimensional environment with a defined medium. Organoids were examined using meibocyte marker PPARγ and ductal cell marker Krt6A. Lipid content was assessed by Nile red oil stain. The differentiated mouse meibomian organoids were spherical in shape (diameter= 88±27µm) and comprised of meibocytes and ductal cells. The central region of the organoids were filled with oil, indicating complete meibocyte maturation. Intriguingly, the Irx3 mutant organoids displayed heterogeneous characteristics. The mutant organoid displayed an enlarged spherical shape (diameter= 162±96µm). Some mutant organoids continued to proliferate in differentiation medium as indicated by positive Ki67 staining. Moreover, some mutant organoids have reduced PPARγ staining, with lipid containing cells identified in the central region. Our results suggest that Irx3 was involved in controlling organoid proliferation and could affect meibocyte maturation. Irx3/5 genes may play a role in maintaining meibocyte homeostasis, and their inactivation may disrupt meibocyte replenishment, leading to atrophic glands and dry eye disease in the mutant animals. Our meibomian gland organoid offers a novel in vitro model for studying the mechanism for meibocyte replenishment and development of therapeutic approaches for managing dry eye disease.
Funding Source: This project is supported by the Health and Medical Research Fund, Hong Kong (HMRF 09203156).
