Abstract
Abstract: :
Purpose:To determine if a population of undifferentiated, actively proliferating, non-adherent cells isolated from human conjunctival tissue are potentially stem cells by examining their ability to self-renew, clonally expand, differentiate into various cell lineages and repair injured tissue. Methods:Conjunctival tissue from a biopsy 21-year-old female was placed in organ culture in RPMI medium supplemented with 10% FBS. Within 10 days of culture, a large non-adherent undifferentiated population of cells was seen in one culture. These cells were removed and further cultured either in RPMI with 1% FBS or 20 ng/ml EGF. Self renewal of cells was performed by seeding a few undifferentiated conjunctival cells into culture flasks and monitoring their ability to repopulate the flask. Clonal analysis consisted of seeding one cell per tissue culture well of a 48-well and tracking its cell division. Undifferentiated conjunctival cells were evaluated by immunocytochemistry for their expression of markers used to identify stem cells in other tissues. The markers used were nestin (intermediate filaments) and Ki-67 (proliferation). Undifferentiated conjunctival cells induced to differentiate by seeding them onto gelatin-coated coverslips were evaluated by immunocytochemistry for their ability to differentiate into various cell lineages including epithelial (MUC 5AC, keratins 4 and 7, UEA-1 and HPA lectins), neural (GFAP, MAP 2, synaptophysin) and muscle (α-smooth muscle actin.). Undifferentiated conjunctival cells were added to wounded human conjunctival fibroblasts (by scraping a portion of cells from the plate) to evaluate their ability to integrate and differentiate into the fibroblast lineage. Results:Undifferentiated conjunctival cells were capable of self renewal by repopulation of their culture vessels. By clonal analysis, one cell was able to give rise to many cells eventually filling the culture well. Most (99%) undifferentiated cells expressed both nestin and Ki-67. However, both nestin and Ki-67 decreased with time in culture of differentiated cells. Differentiated cells expressed the epithelial markers MUC 5AC, keratins 4 and 7, and the lectins UEA-1 and HPA . They were positive for the neural marker synaptophysin, but not for GFAP, MAP2 nor α-smooth muscle actin. When undifferentiated conjunctival cells were added to injured fibroblast monolayer, within 2 days a few cells adhered to the dish and began to assume a fibroblast morphology. Conclusion:Adult human conjunctival tissue is endowed with a population of cells that can be isolated and exhibit many characteristics of stem cells.
Keywords: 365 conjunctiva • 370 cornea: basic science • 434 immunohistochemistry