Mechanisms of Dedifferentiation of Adult Human Retinal Pigment Epithelial Cells in vitro. Morphological and Molecular Genetic Analysis

Abstract

Changes in morphology and molecular genetic profile of human retinal pigment epithelial (RPE) cells in vitro were studied when exposed to the basic fibroblast growth factor (bFGF). The cell morphology was estimated by cell area and perimeter, spreading and polarization coefficients. It was demonstrated that the number of elongated (fibroblast-like) cells and the number of cells, which size was less than in the control increased in 48 h after adding the factor to the culture. At the same time, the cell proliferative activity decreased (according to MTT test). Immunocytochemical analysis demonstrated a decrease in the staining for connexin Cx43 and an increase in the intensity of staining for protein the Otx2 neuroepithelium protein. Simultaneously, the number of nestin-positive cells and βIII-tubulin positive cells increased. Using quantitative real-time PCR method, an increase in mRNA expression of the KLF4, OCT4, NANOG, OTX2, and NES and decrease in mRNA expression of the MITF and KRT18 were detected in RPE cells treated with bFGF; this indicates enhancement of cell dedifferentiation. These data are confirmed by a decrease in the COL1A1 mRNA expression indicating a decrease in synthetic cell activity. The results indicate that single (short-time) effect by bFGF is sufficient to activate the mechanism, which decreases the cell differentiation level toward neuroepithelium.