Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran.
Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran.
Exp Eye Res. 2021 Jan;202:108346. doi: 10.1016/j.exer.2020.108346. Epub 2020 Nov 2.
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.
视网膜退行性疾病被认为是全世界的一个主要挑战，而干细胞疗法是一种有前途的方法，可以恢复由于 RD 而退化的细胞。MSC 是一种存在于多种组织中的多能干细胞。它们能够分化为各种视网膜细胞类型，因此可以成为各种退行性疾病（如视网膜变性）的良好候选者。β-胡萝卜素是一种抗氧化剂，可以在使用适当支架的情况下加速干细胞分化。在这项研究中，我们评估了β-胡萝卜素对从鼠眼睫状上皮分离的 MSC 的分化潜能的影响，这些 MSC 被培养在含有 10% FBS 的 DMEM 培养基中，并通过检测其表面抗原来鉴定。我们制备了三种 3D 培养系统，即藻酸盐、藻酸盐/明胶和明胶水凝胶，并通过 SEM 检查了它们的结构。将 MSC 培养在 3D 和 2D 培养系统支架上，然后用含有 50μM β-巯基乙醇、1×非必需氨基酸最小培养基和 20%无血清替代物和β-胡萝卜素的分化培养基处理。通过 MTT 和 ICC 分别检查 MSC 的活力和分化能力。还通过 qPCR 评估了几种视网膜特异性基因（Nestin、RPE65 和 Rhodopsin）的表达变化。从鼠眼睫状上皮分离的细胞中，超过 80%的细胞呈 MSC 特异性标志物阳性。在所有基于藻酸盐的支架上生长的 MSC 的活力率均高于 70%。与 2D 培养相比，在含有β-胡萝卜素的分化培养基中培养的基于藻酸盐的支架上的 MSC 表达更高水平的视蛋白蛋白。此外，在基于藻酸盐的支架上生长的经β-胡萝卜素处理的 MSC 中，Nestin、Rhodopsin 和 RPE65 基因的表达上调。我们的结果表明，在分化培养基中加入β-胡萝卜素，并应用基于藻酸盐的支架，可以诱导鼠眼睫状上皮来源的 MSC 向特化的视网膜细胞分化。
