Polarized Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cell Monolayers Have Higher Resistance to Oxidative Stress-Induced Cell Death Than Nonpolarized Cultures

Abstract

Oxidative stress-mediated injury to the retinal pigment epithelium (RPE) is a major factor involved in the pathogenesis of age-related macular degeneration (AMD), the leading cause of blindness in the elderly. Human embryonic stem cell (hESC)-derived RPE cells are currently being evaluated for their potential for cell therapy in AMD patients through subretinal injection of cells in suspension and subretinal placement as a polarized monolayer. To gain an understanding of how transplanted RPE cells will respond to the highly oxidatively stressed environment of an AMD patient eye, we compared the survival of polarized and nonpolarized RPE cultures following oxidative stress treatment. Polarized, nonpolarized/confluent, nonpolarized/subconfluent hESC-RPE cells were treated with H 2 O 2 . Terminal deoxynucleotidyl transferase dUTP nick end labeling stains revealed the highest amount of cell death in subconfluent hESC-RPE cells and little cell death in polarized hESC-RPE cells with H 2 O 2 treatment. There were higher levels of proapoptotic factors (phosphorylated p38, phosphorylated c-Jun NH 2 -terminal kinase, Bax, and cleaved caspase 3 fragments) in treated nonpolarized RPE-particularly subconfluent cells-relative to polarized cells. On the other hand, polarized RPE cells had constitutively higher levels of cell survival and antiapoptotic signaling factors such as p-Akt and Bcl-2, as well as antioxidants superoxide dismutase 1 and catalase relative to nonpolarized cells, that possibly contributed to polarized cells' higher tolerance to oxidative stress compared with nonpolarized RPE cells. Subconfluent cells were particularly sensitive to oxidative stress-induced apoptosis. These results suggest that implantation of polarized hESC-RPE monolayers for treating AMD patients with geographic atrophy should have better survival than injections of hESC-RPE cells in suspension. STEM CELLS TRANSLATIONAL MEDICINE 2015;4:10-20

Inhibition of DNA Methylation and Methyl-CpG-Binding Protein 2 Suppresses RPE Transdifferentiation: Relevance to Proliferative Vitreoretinopathy

“…45 Retinal pigment epithelium cells were plated on a permeable membrane insert (12-mm diameter, 0.4-mm pore size; Transwell; Costar, Cambridge, MA, USA), and cells were cultured for at least 1 month to form differentiated monolayers with a stable transepithelial resistance (TER) of >350 ohms/cm 2 . Transepithelial resistance was measured using an EVOM volt-ohm meter (World Precision Instruments, Sarasota, FL, USA).…”

An Innovative Surgical Technique for Subretinal Transplantation of Human Embryonic Stem Cell-Derived Retinal Pigmented Epithelium in Yucatan Mini Pigs: Preliminary Results

“…The advantages of the surgically foldable MSPS carrier include customized macular shape in diameter and thickness [8], ultrathin parts with diffusion [7], and excellent RPE adherence [10,11] and survival [12], whereas its nondegradability and unknown long-term effects can be seen as disadvantages. The study criteria for successful implantation were appropriate placement of the implant in the subretinal space, as assessed by OCT and post-mortem histology.…”

“…MSPMs that did not meet the quality control criteria were discarded. This process generated a polarized hESC-RPE monolayer with characteristics similar to human fetal RPE which would promote survival of the monolayer in vivo [8,11].…”

Progressing a human embryonic stem-cell-based regenerative medicine therapy towards the clinic

“…While at high cell densities they are able to redifferentiate to RPE, at lower cell densities they maintain a mesenchymal phenotype, lacking any RPE functionality [18,19]. Additionally, RPE monolayers have been shown to be more resistant to oxidative stress-induced cell death [20]. The additional concern is that the Bruchs membrane, upon which the native RPE monolayer sits, is compromised in the aged eye and the disease state, and hESC-derived RPE have been shown to adhere very poorly [20].…”

“…Additionally, RPE monolayers have been shown to be more resistant to oxidative stress-induced cell death [20]. The additional concern is that the Bruchs membrane, upon which the native RPE monolayer sits, is compromised in the aged eye and the disease state, and hESC-derived RPE have been shown to adhere very poorly [20]. Indeed, RPE cells transplanted into preclinical models as a monolayer survive better than those implanted as a dissociated suspension [21].…”