Abstract
Purpose: :
Age–related macular degeneration (AMD), the most common cause of irreversible vision loss in the elderly, results from oxidative damage to the retinal pigment epithelium (RPE) and concomitant photoreceptor cell apoptosis due to loss of RPE trophic support. In spite of the availability of a number of therapeutic measures proposed for AMD, including transplantation of embryonic stem (ES) cells into the retina, the treatment of AMD remains unsatisfactory. Stem cell transplantation mostly involves driving the uncommitted ES cells to differentiate and replace degenerating neurons. Recent studies suggesting transplanted stem cells as a source of trophic factors in a variety of tissues led us to investigate the potential of stem cell–derived factors in protecting RPE cells from oxidative damage.
Methods: :
Human retinal progenitor cells (hRPCs) isolated from donor neonatal retinas were expanded in vitro and characterized for stemness using RT–PCR and immunological assays. The effects of oxidative damage on 2 human RPE cell lines (ARPE–19 and BHR cells) were investigated in the presence or absence of known growth factors or 48h–conditioned media (CM) from hRPCs under intense light exposure or a hydrogen peroxide–generating system utilizing glucose oxidase/glucose. Cell survival was determined by MTS assay and fluorescent dyes (calcein AM and ethidium homodimer); while JC–1 was used to determine mitochondrial membrane integrity.
Results: :
Cultured hRPCs rapidly proliferated and formed ball–like clusters (neurospheres) in the presence of growth factors such as fibroblast growth factor–2 and epidermal growth factor. Immunoblotting and RT–PCR analysis showed expression of retinal progenitor cell markers including nestin, pax6, and neurofilament 68 in hRPCs. Consistent with their lack of differentiation, hRPCs had low levels of mature retinal cell markers using antibodies against opsin (photoreceptors), glial fibrillary acidic protein or glutamine synthetase (retinal glia), and CD31 (endothelial cells). Unlike, other known growth factors or neurotrophins, CM from hRPCs distinctly inhibited RPE cell death and collapse of mitochondrial membrane potential due to oxidative damage.
Conclusions: :
Our study shows that human progenitor cells isolated from neonatal retinas secrete anti–apoptotic molecules that can protect the RPE from oxidative damage in vitro. The putative hRPC–derived trophic factors could prove useful in the treatment of AMD.
Keywords: neuroprotection • retinal pigment epithelium • oxidation/oxidative or free radical damage