In vitro Derivation of Polarized hES-RPE Cells and Their Effects on Retinal Progenitor Cells

Abstract

Purpose: : Dysfunction and/or death of RPE play a critical role in the pathogenesis of age-related macular degeneration (AMD). There are as yet no effective treatments for the late form of dry AMD. The purpose of this study is to develop a novel procedure to induce the differentiation of hES cells into large numbers of functionally polarized hES-RPE cells suitable for subretinal transplantation in animal models and to investigate their effects on human retinal progenitor cells.

Methods: : 1). Human embryonic stem cells 3 (hES-3) were used for derivation into RPE-like cells The hES-3 cell aggregates were first kept in suspension culture in neural differentiation medium. The neural precursors were then plated on the gelatin or laminin coated cell culture dishes with RPE differentiation medium for further differentiation into putative RPE-like cells. 2). The human retinal progenitor cell (RPC) growth and differentiation assay was conducted by culturing RPC on fibronectin coated cell culture plates with hES-RPE or fetal RPE conditioned medium, or RPC medium plus 50ng/ml recombinant pigment epithelium derived factor (PEDF). 3). RT-PCR and Western blot were used to verify the expression of RPE specific genes. 4). ELISA was performed to measure PEDF levels from the conditioned or non-conditioned RPC culture mediums.

Results: : The putative pigmented hexagonal RPE cells appeared as early as 4 weeks in our culture conditions and reached high enough cell numbers for subculture at about 8 weeks. The hES-RPE cells expressed high levels of RPE-specific gene products including RPE65, VMD2, PEDF and CRELBP as shown by RT-PCR, Western blot and ELISA analyses. hES-RPE conditioned medium contained high levels of PEDF. Increased numbers of RPC cells were observed when the RPC were cultured with 50% polarized hES-RPE conditioned medium or with 50 ng/ml recombinant PEDF, suggesting that hES-RPE support and protect retinal cells through their secreted high level of PEDF.

Conclusions: : We successfully induced the differentiation of hES-3 cells into functionally polarized hES-RPE cells that exhibit the characteristics similar to those of native RPE. These hES-RPE cells can be expanded in high yield and purity, and secrete a large amount of PEDF that can support RPC.

Keywords: age-related macular degeneration • retinal degenerations: cell biology • retinal pigment epithelium