Dong Chunxiao, Zou Dulei, Duan Haoyun, Hu Xiangyue, Zhou Qingjun, Shi Weiyun, Li Zongyi
Department of Medicine, Qingdao University, Qingdao, 266071, China.
Eye Institute of Shandong First Medical University, State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Qingdao, 266071, China.
Eye Vis (Lond). 2023 Aug 2;10(1):34. doi: 10.1186/s40662-023-00351-4.
Stem cell therapy is a promising strategy for the treatment of corneal endothelial dysfunction, and the need to find functional alternative seed cells of corneal endothelial cells (CECs) is urgent. Here, we determined the feasibility of using the retinal pigment epithelium (RPE) as an equivalent substitute for the treatment of corneal endothelial dysfunction.
RPE cells and CECs in situ were obtained from healthy New Zealand male rabbits, and the similarities and differences between them were analyzed by electron microscopy, immunofluorescent staining, and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Rabbit primary RPE cells and CECs were isolated and cultivated ex vivo, and Na+/K+-ATPase activity and cellular permeability were detected at passage 2. The injection of cultivated rabbit primary RPE cells, CECs and human embryonic stem cell (hESC)-derived RPE cells was performed on rabbits with corneal endothelial dysfunction. Then, the therapeutic effects were evaluated by corneal transparency, central corneal thickness, enzyme linked immunosorbent assay (ELISA), qRT-PCR and immunofluorescent staining.
The rabbit RPE cells were similar in form to CECs in situ and ex vivo, showing a larger regular hexagonal shape and a lower cell density, with numerous tightly formed cell junctions and hemidesmosomes. Moreover, RPE cells presented a stronger barrier and ionic pumping capacity than CECs. When intracamerally injected into the rabbits, the transplanted primary RPE cells could dissolve corneal edema and decrease corneal thickness, with effects similar to those of CECs. In addition, the transplantation of hESC-derived RPE cells exhibited a similar therapeutic effect and restored corneal transparency and thickness within seven days. qRT-PCR results showed that the expressions of CEC markers, like CD200 and S100A4, increased, and the RPE markers OTX2, BEST1 and MITF significantly decreased in the transplanted RPE cells. Furthermore, we have demonstrated that rabbits transplanted with hESC-derived RPE cells maintained normal corneal thickness and exhibited slight pigmentation in the central cornea one month after surgery. Immunostaining results showed that the HuNu-positive transplanted cells survived and expressed ZO1, ATP1A1 and MITF.
RPE cells and CECs showed high structural and functional similarities in barrier and pump characteristics. Intracameral injection of primary RPE cells and hESC-derived RPE cells can effectively restore rabbit corneal clarity and thickness and maintain normal corneal function. This study is the first to report the effectiveness of RPE cells for corneal endothelial dysfunction, suggesting the feasibility of hESC-derived RPE cells as an equivalent substitute for CECs.
干细胞疗法是治疗角膜内皮功能障碍的一种有前景的策略，因此迫切需要找到角膜内皮细胞（CECs）的功能性替代种子细胞。在此，我们确定了使用视网膜色素上皮（RPE）作为等效替代物治疗角膜内皮功能障碍的可行性。
从健康的新西兰雄性兔获取原位RPE细胞和CECs，并通过电子显微镜、免疫荧光染色和定量实时逆转录聚合酶链反应（qRT-PCR）分析它们之间的异同。体外分离并培养兔原代RPE细胞和CECs，在第2代检测Na+/K+-ATP酶活性和细胞通透性。对角膜内皮功能障碍的兔注射培养的兔原代RPE细胞、CECs和人胚胎干细胞（hESC）来源的RPE细胞。然后，通过角膜透明度、中央角膜厚度、酶联免疫吸附测定（ELISA）、qRT-PCR和免疫荧光染色评估治疗效果。
兔RPE细胞在原位和体外的形态与CECs相似，呈较大的规则六边形，细胞密度较低，有许多紧密形成的细胞连接和半桥粒。此外，RPE细胞比CECs具有更强的屏障和离子泵浦能力。当眼内注射到兔体内时，移植的原代RPE细胞可消除角膜水肿并降低角膜厚度，效果与CECs相似。此外，hESC来源的RPE细胞移植表现出类似的治疗效果，并在7天内恢复角膜透明度和厚度。qRT-PCR结果显示，移植的RPE细胞中CEC标志物如CD200和S100A4的表达增加，而RPE标志物OTX2、BEST1和MITF显著降低。此外，我们证明，移植hESC来源的RPE细胞的兔在术后1个月保持正常角膜厚度，中央角膜出现轻微色素沉着。免疫染色结果显示，HuNu阳性的移植细胞存活并表达ZO1、ATP1A1和MITF。
RPE细胞和CECs在屏障和泵浦特性方面表现出高度的结构和功能相似性。眼内注射原代RPE细胞和hESC来源的RPE细胞可有效恢复兔角膜的清晰度和厚度，并维持正常角膜功能。本研究首次报道了RPE细胞治疗角膜内皮功能障碍的有效性，提示hESC来源的RPE细胞作为CECs等效替代物的可行性。
