Human Retinal Pigmented Epithelium cells (hRPE) Potential for Retinal Regeneration Induction Trough Simulating Retinal Vascular Membrane (RVM) Using Fibrin Sealant Scaffold

Retina regeneration through reprogramming process is an interesting topic for the retinal disorders and regeneration study. This phenomenon naturally happens in a few of amphibians even if the neural retina is completely removed by surgery. Studies show the critical role of FGF2 factor and RVM microenvironment in retinal regeneration. Thus, this study pays particular attention to regeneration potential of human RPE cell under the influence of the fibrin glue 3D structure as FGF2 reservoir and RVM like space.

hRPE cells were isolated from donor eyes of two adult humans. The cells were encapsulated with three concentrations of fibrin glue (FG1:42mg/dl, FG2:84mg/dl, FG3:126mg/dl) and FBS treated with 20% concentration was considered as control. Gene expressions of PAX6, MMP2, RPE65, ACTA2, CRX, Thy-1, NogoA, and Integrin-B1 were analyzed, using real-time PCR.

Gene expression of Integrin B1 and Thy-1 were significantly higher in FG1-treated cells compared to controls. Also, PAX6 gene expression in FG1 concentration compared to control and FG3 showed a significant increase, while expression of MMP2 gene expression at FG1 concentration showed a higher expression level than other concentrations, but only significant compared to FG3. RPE65 expression in FG1, FG2 concentrations shows a significant increase compared to control; ACTA2 gene expression was lower than 3 concentrations compared to control, but it was not significant. Also, expression of NogoA gene showed a significant decrease in FG1 concentration compared to control and FG3. In addition, the expression of the CRX gene was negative in all treatments and control. The immunocytochemistry results indicate that PAX6 protein is not expressed in incubated specimens and controls in adult RPE cells, but these cells express specific proteins such as RPE65, cytokeratin8.18 in both control and incubated specimens are positive.

Adult RPE cells at the concentration of FG1 (42 mg/dl) maintain certain adult characteristics, but exhibited signs of multipotency, which could indicate the potential of these cells for retinal tissue regeneration. This effect appears to be due to the presence of adhesion molecules such as Fibronectin and Laminin in fibrin glue that simulates the RVM like space, as well as the creation of an FGF2 reservoir and its localization, which leads to an increase of the PAX6 gene expression.