Generation of Retinal Pigmented Epithelium-Like Cells from Pigmented Spheres Differentiated from Bone Marrow Stromal Cell-Derived Neurospheres.

Abstract

Retinal degeneration causes blindness, and cell replacement is a potential therapy. The purpose of this study is to formation of pigmented neurospheres in a simple medium, low-cost, high-performance manner over a short period of time while expressing markers of RPE cells and the activation of specific genes of the pigment cells. Also, these neurospheres have the ability to produce a monolayer of retinal pigment epithelium-like cells (RPELC) with the ability of photoreceptor outer segment phagocytosis. BMSC were isolated from pigmented hooded male rats and were immunoreactive to BMSC markers, then converted into neurospheres, differentiated into pigmented spheres (PS), and characterized using Retinal pigment epithelium-specific 65kDa protein (RPE65), Retinaldehyde-binding protein 1 (CRALBP) and orthodenticle homeobox 2 (OTX2) markers by immunocytochemistry, RT-PCR and RT-qPCR. The PS were harvested into RPELC. The functionality of RPELC was evaluated by phagocytosis of fluorescein-labeled photoreceptor outer segment. The BMSC immunophenotype was confirmed by immunostained for fibronectin, CD90, CD166 and CD44. These cells differentiated into osteogenic and lipogenic cells. The generated neurospheres were immunoreactive to nestin and stemness genes. The PS after 7-14days were positive for RPE65 (92.76-100%), CRALBP (95.21-100%) and OTX2 (94.88-100%), and after 30days RT-PCR, qPCR revealed increasing in gene expression. The PS formed a single layer of RPELC after cultivation and phagocyte photoreceptor outer segments. Bone marrow stromal stem cells can differentiate into functional retinal pigmented epithelium cells in a simple, low-cost, high-performance manner over a short period of time. These cells due to expressing the RPELC genes and markers can be used in cell replacement therapy for degenerative diseases including age-related macular degeneration as well as retinitis pigmentosa.