Pyruvate Enhances the Differentiation Profile of the Human Retinal Pigment Epithelium Cell Line ARPE-19

Abstract

Purpose: : Transplantation of the retinal pigment epithelium (RPE) requires cells that are phenotypically close to native RPE. Cultured RPE may become an option for transplantation in RPE degenerative disease such as age-related macular degeneration. However RPE undergo de-pigmentation and lose markers of differentiation when grown in culture. Pyruvate is a potent intracellular buffer and a scavenger of hydrogen peroxide. Many studies suggest pyruvate is protective against oxidative stress and other stressors in the retina. We are proposing a simple culture protocol that promotes differentiation markers such as CRALBP, RPE65 and dense pigmentation in higher passages of ARPE-19 cells when pyruvate is added to culture medium. This is the first study to demonstrate significant RPE65 expression by western blot analysis in the human RPE cell line ARPE-19.

Methods: : Passages p22 - p28 of the human RPE cell line ARPE-19 were grown on polyester filters in either DMEM/F12, DMEM with or without pyruvate and foetal calf serum concentration of 1% for up to 3 months. Cultures were fed twice a week. Pigmentation, immunocytochemistry, apical-basal polarity, VEGF secretion and western blots were used to assess differentiation.

Results: : Pyruvate accelerated pigmentation in combination with DMEM both at low (1g/L) and high (4.5g/L) glucose concentration. CRALBP and RPE65 expression is significantly increased by pyruvate as shown by western blot analysis. Pyruvate supplementation induced both quicker onset of circumferential actin distribution as well as suppression of cytokeratin 8 (CK 8) irrespective of glucose concentration. Immune positivity of CK 8 was found to be inversely related to pigmentation and pmel17 expression as shown by confocal microscopy. VEGF secretion was not affected by pyruvate.

Conclusions: : Pyruvate is a valuable growth media component whose beneficial effect on ARPE-19 is demonstrated by promoting multiple differentiation characteristics. The exact mechanism of pyruvate action on RPE differentiation is unknown but maybe due to its potent buffering capacity and anti-oxidative properties. It is unlikely that pyruvate acts simply as an additional energy source since favourable effects occurred both at low and high concentrations of glucose.

Keywords: retinal pigment epithelium • age-related macular degeneration • differentiation