Abstract
The pigmented ciliary epithelium (PCE) of mammalian eye harbors resident population of stem cells that lie in apposition with endothelial cells which release vascular endothelial growth factor (VEGF) that may influence the fate and function of these stem cells in ways that remain unclear. We examined the role of VEGF in proliferation of PCE stem cells and expression of Notch, Jagged, N-Cadherin and β-Catenin which are known to maintain proliferation state of neural stem cells. We cultured human PCE cells obtained from 12-20 weeks old fetal eyes. The neurospheres were analyzed for the proliferation capacity of PCE stem cells in presence of VEGF on 3,6 and 9 day. Real time PCR was used to quantitate the mRNA expression of above mentioned genes on PCE derived neurospheres on 3,6 and 9 day. We found increased number of neurospheres when PCE stem cells were stimulated with VEGF alongwith epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) than EGF and bFGF. BrdU immunostaining was done to analyze the proliferation of CE cells and presence of neural and retinal progenitor markers such as Nestin and Pax6 were also investigated. An increased Notch and Jagged mRNA was observed on 6th day in VEGF, EGF and bFGF treated PCE cells as compared to 0,3 and 9 day. A similar pattern was noticed with N-cadherin and β-catenin mRNA levels. These findings may clarify the role of VEGF on PCE stem cell proliferation with possible involvement of Notch, Jagged, N-cadherin and β-Catenin. The data may suggest importance of harvesting 6th day neurospheres for transplantation purposes in preclinical investigations pertaining to retinal degenerative diseases, however, additional studies are needed to substantiate the findings.
Keywords: Basic fibroblast growth factor, β catenin, ciliary epithelium, epidermal growth factor, jagged, N cadherin, notch, vascular endothelial growth factor