Effect of β-carotene on the differentiation potential of ciliary epithelium-derived MSCs isolated from mouse eyes on alginate-based scaffolds

Abstract

Retinal degenerative diseases are considered a major challenge all over the world, and stem cell therapy is a promising approach to restore degenerative cells due to RD. MSCs are multipotent stem cells found in a variety of tissues. They are capable of differentiating into various retinal cell types, so it can be a good candidate for various degenerative disorders like retinal degenerations. β-carotene is an antioxidant that could accelerate the stem cell differentiation while using the proper scaffold. In this study, we evaluated the effect of β-carotene on the differentiation potential of ciliary epithelium-derived MSCs isolated from mouse eyes on alginate-based scaffolds. MSCs were isolated from mouse ciliary epithelium, cultured in DMEM medium supplemented with 10% FBS, and identified by detecting their surface antigens. Three 3D culture systems, alginate, alginate/gelatin, and gelatin hydrogels were prepared, and their structures were checked via SEM. MSCs were cultured on 3D and 2D culture system scaffolds following treated with differentiation medium containing 50 μM β-mercaptoethanol, 1 × minimum essential medium–nonessential amino acids and 20% of knockout serum replacement and β-carotene. MSCs viability and differentiation ability were examined by MTT and ICC, respectively. The expression changes of several retinal specific genes (Nestin, RPE65, and Rhodopsin) were also evaluated by qPCR. Over 80% of cells isolated from mouse ciliary epithelium were positive for MSC-specific markers. The viability rates of MSCs grown on all alginate-based scaffolds were above 70%. MSCs cultured on alginate-based scaffold in the differentiation medium containing β-carotene expressed higher levels of rhodopsin protein compared to a 2D culture. Also, the expressions of Nestin, Rhodopsin, and RPE65 genes were upregulated in β-carotene-treated MSCs grown on alginate-based scaffolds. Our results indicate that the addition of β-carotene to the differentiation medium, along with applying alginate-based scaffolds, could induce higher differentiation in mouse ciliary epithelium-derived MSCs into specialized retinal cells.