Abstract
Purpose :
We have reported that transplanted trabecular meshwork stem cells (TMSC) can reduce intraocular pressure (IOP) in a mouse glaucoma model. In this study, we hypothesized that TMSC and TM cells (TMC) can regulate the Schlemm’s canal endothelial cell (SCEC) permeability which contributes for IOP regulation.
Methods :
Cultured human TMSC, TM and SCEC were verified using flow cytometry, immunostaining, qPCR and Western blotting. TMSC or TMC were co-cultured with SCEC using culture inserts in 6-well or 24-well plates without contacting to each other. The effects of condition media (CM) from TMSC or TMC were also examined for SCEC culture. Transendothelial electrical resistance (TEER) was measured daily for 7 days to assess SCEC permeability. SCEC treated with Rho kinase inhibitor Y27632 and without any treatments served as controls. At least 3 SCEC strains from different donors were used as biological repeats. Images were analyzed using ImageJ and Prism. P<0.05 was considered statistically significant.
Results :
TMSC expressing more than 90% of CD73, CD90, CD105 by flow cytometry and positive to OCT4 by staining were used for the experiments. TMC were confirmed by increased myocilin expression after dexamethasone treatment. SCEC were stained positive to Prox-1, VE-cadherin (VE-Cad), and fibulin-2. SCEC had significantly increased expression of Prox-1 (10-15 times), CD31 (5-6 times), VE-Cad (60-80 times), ZO-1 (5-6 times), fibulin-2 (>1000 times), and similar or reduced expression of connexin 43 and N-Cad, as compared to TMC. SCEC co-cultured with TMSC cells or with TMSC-CM started to show reduced TEER from day 2 after co-culture. TEER reduced to about 30% of that of SCEC cultured along at day 7, comparable to the effect of Y27632. SCEC co-cultured with TMC or TM-CM showed similar TEER to SCEC cultured alone. Immunostaining showed that VE-Cad expression in the SCEC with TMC was similar to that of SCEC cultured alone, which was significantly reduced in the SCEC with TMSC or with Y27632. The result indicates that VE-Cad plays a very important role in regulating the permeability of SCEC.
Conclusions :
Human TMSC and TMSC-CM were able to reduce the permeability of co-cultured SCEC, while TMC had little effect on the SCEC permeability. It reveals that TMSC might secret important factors that can regulate SCEC permeability while TMC might need to be in contact with SCEC to regulate SCEC function.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.