Abstract
Purpose :
Conjunctiva maintains the ocular surface health by lubrication and protects the eye ball from the external environments. Conjunctival stem cell is a bipotent progenitor that differentiates into conjunctival epithelium and goblet cell. Wnt/β-catenin signaling has been known to play an important role in regulation of stem cell proliferation, differentiation, and maintenance. The role of the Wnt/β-catenin signaling in conjunctival stem cell has not understood. This study investigated the effect of Wnt/β-catenin signaling on conjunctival stem cells.
Methods :
For in vivo analysis, C57BL/6J mouse were used. Mouse cornea and conjunctival epithelium was removed by alcohol delamination using 3mm corneal trephine. Experiment group was injected with BrdU and then treated with CHIR, which activates Wnt/β-catenin signaling. Conjunctiva was immunostained with the p63a as a conjunctival stem cell marker and BrdU. Real-time PCR was adopted to investigate the changes in Wnt/β-catenin related-genes. Human conjunctiva tissue was cultured for in vitro analysis. Conjunctival stem cells were isolated from explanted cells and seeded on growth-arrest NIH 3T3 feeder layer. Changes in stem cell markers were determined after CHIR treatment. Cultured stem cells were placed in differentiation media condition before or after CHIR treatment to find the expression of goblet cell marker K7.
Results :
In mouse epithelial wound model, double immunofluorescence revealed that both p63a and BrdU intensity were increased compared with un-wounded mouse. Real-time PCR analysis showed that the Wnt2b and Wnt3a were upregulated. The topical application of CHIR induced p63a and BrdU double positive cells in wounded mouse. CHIR treatment increased the expression of stem cell marker ABCG2 and DN-p63a in both primary cultured explant cells and isolated conjunctival stem cells compared to negative control. The number of K7-positive cells was increased in the cultures under differentiation condition, in which cells were cultured with CHIR prior to differentiation. However, when we treated cells with CHIR under differentiation condition, no difference in the number of K7-positive cells was observed between control-and CHIR-treated cultures.
Conclusions :
These findings suggest that the activation of Wnt/β-catenin signaling pathway promotes self-renewal and stemness regulation of conjunctival stem cells.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.