Separation of Phenotypically Distinct Subpopulations of Cultured Human Retinal Pigment Epithelial Cells

Abstract

Like most epithelial cells that are isolated from tissues and placed in culture, the phenotype of human retinal pigment epithelial cells (RPE) in vitro is more variable than for the same cells in situ. The phenotypic heterogeneity of the cultures has been attributed to varying stages of differentiation of the cells induced by the culture environment. In this study we show that within the same cultures RPE cells exist as phenotypic variants which are distinct and stable. Two phenotypically distinct subpopulations were identified, one epithelioid and one fusiform, that were present from the first spreading event in primary culture through multiple serial passages and when maintained for extended periods at confluency. Cell aggregation studies indicated differences in cellular adhesions, known determinants of cell shape, between the subpopulations. Methods to separate the subpopulations were developed which are based on the differential trypsin sensitivity of adhesions. The separated subpopulations had the same RPE cytokeratins by immunoblotting, but cytokeratin filaments (and actin filaments) had different organizations. The study indicates that RPE cell cultures contain at least two subpopulations of phenotypically distinct cells under identical culture conditions that can be separated and propagated independently. The phenotypic variants offer a model system for investigating determinants of epithelial cell shape in RPE. Further, the separation methods might be applied to test for phenotypic variants in other types of cultured epithelia.