Retinal pigment epithelium (RPE) differentiated from human induced pluripotent stem cells, called induced retinal pigment epithelium (iRPE), is being explored as a cell-based therapy for the treatment of retinal degenerative diseases, especially age-related macular degeneration. The success of RPE implantation is linked to the use of biomimetic scaffolds that simulate Bruch's membrane and promote RPE maturation and integration as a functional tissue. Due to difficulties associated with animal protein-derived scaffolds, including sterility and pro-inflammatory responses, current practices favor the use of synthetic polymers, such as polycaprolactone (PCL), for generating nanofibrous scaffolds. In this study, we tested the hypothesis that plant protein-derived fibrous scaffolds can provide favorable conditions permissive for the maturation of RPE tissue sheets in vitro. Our natural, soy protein-derived nanofibrous scaffolds exhibited a J-shaped stress–strain curve that more closely resembled the mechanical properties of native tissues than PCL with significantly higher hydrophilicity of the natural scaffolds, favoring in vivo implantation. We then demonstrate that iRPE sheets growing on these soy protein scaffolds are equivalent to iRPE monolayers cultured on synthetic PCL nanofibrous scaffolds. Immunohistochemistry demonstrated RPE-like morphology and functionality with appropriate localization of RPE markers RPE65, PMEL17, Ezrin, and ZO1 and with anticipated histotypic polarization of vascular endothelial growth factor and pigment epithelium-derived growth factor as indicated by enzyme-linked immunosorbent assay. Scanning electron microscopy revealed dense microvilli on the cell surface and homogeneous tight junctional contacts between the cells. Finally, comparative transcriptome analysis in conjunction with principal component analysis demonstrated that iRPE on nanofibrous scaffolds, either natural or synthetic, matured more consistently than on nonfibrous substrates. Taken together, our studies suggest that the maturation of cultured iRPE sheets for subsequent clinical applications might benefit from the use of nanofibrous scaffolds generated from natural proteins.

中文翻译：

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大豆蛋白纳米纤维支架用于人诱导多能干细胞衍生的视网膜色素上皮的均匀成熟。

从人类诱导性多能干细胞分化而来的视网膜色素上皮 (RPE)，称为诱导性视网膜色素上皮 (iRPE)，正在探索作为一种基于细胞的疗法，用于治疗视网膜退行性疾病，尤其是与年龄相关的黄斑变性。RPE 植入的成功与使用仿生支架有关，该支架模拟布鲁赫膜并促进 RPE 成熟和整合为功能组织。由于与动物蛋白衍生支架相关的困难，包括不育和促炎反应，目前的做法倾向于使用合成聚合物，如聚己内酯 (PCL)，用于生成纳米纤维支架。在这项研究中，体外。我们的天然大豆蛋白衍生纳米纤维支架表现出 J 形应力-应变曲线，与天然支架的亲水性显着更高的 PCL 相比，更接近于天然组织的机械特性，有利于体内植入。然后我们证明在这些大豆蛋白支架上生长的 iRPE 片相当于在合成 PCL 纳米纤维支架上培养的 iRPE 单层。免疫组织化学证明了 RPE 样形态和功能，RPE 标记物 RPE65、PMEL17、Ezrin 和 ZO1 的适当定位以及血管内皮生长因子和色素上皮衍生生长因子的预期组织型极化，如酶联免疫吸附试验所示。扫描电子显微镜显示细胞表面有致密的微绒毛和细胞之间均匀紧密的连接接触。最后，结合主成分分析的比较转录组分析表明，天然或合成的纳米纤维支架上的 iRPE，比在非纤维基质上更稳定地成熟。总之，我们的研究表明，用于后续临床应用的培养 iRPE 片的成熟可能受益于使用由天然蛋白质产生的纳米纤维支架。