Abstract
Purpose: :
Glaucoma is a leading cause of blindness. Reduced cellularity within the trabecular meshworm (TM) is observed with age and correlates with increased outflow resistance and elevated IOP, important risk factors for glaucoma. Cell-based therapy of TM for restoration of aqueous outflow has not been fully explored. Such therapy would require cells capable of assuming a TM phenotype. This study aimed to identify and culture stem cells from TM and to explore the potential of such cells to differentiate into functional TM cells.
Methods: :
Human cornea-scleral tissues were immunostained as whole-mounts using stem cell markers: ABCG2, PAX6, AnkyrinG, Nestin; or TM cell markers: Aquaporin1, MGP, NCAM, CHI3L1, NCAM, and TIMP3. Fresh TM was dissected for explant culture and propagated at low cell density to promote stem cell expansion. Immunofluorescent staining, qPCR, and immuoblotting were used to assess cell phenotypes. Side-population cells were isolated by fluorescent-activated cell sorting using DyeCycle Violet reagent. TM stem cells were induced to differentiate to TM using aqueous humor or fetal bovine serum. Differentiation was confirmed by expression of TM-specific proteins and phagocytic function.
Results: :
Cells immunostaining for stem cell proteins were not limited to the insert region but found throughout the TM. Passaged TM cells contained a side-population sensitive to fumitremorgin C which grew clonally, exhibiting a stem cell phenotype. Expanded stem cells maintained stem cell phenotype but lost TM-specific gene expression. Stem cells cultured in aqueous humor or fetal bovine serum lost stem cell marker expression and markedly upregulated TM tissue-specific gene expression. Phagocytic function also increased as the cell differentiated to TM.
Conclusions: :
Stem cells are found distributed throughout human TM. These cells are distinct from differentiated TM cells, can be isolated as a side population using cell sorting, and can be expanded in culture maintaining their stem cell phenotype. These TM stem cells can be induced to express differentiated TM marker genes and exhibit phagocytic activity typical of TM cells.
Keywords: trabecular meshwork • flow cytometry • differentiation