State Key laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou, China.
PLoS One. 2011;6(11):e25713. doi: 10.1371/journal.pone.0025713. Epub 2011 Nov 11.
Severe ocular surface diseases are some of the most challenging problems that the clinician faces today. Conventional management is generally unsatisfactory, and the long-term ocular consequences of these conditions are devastating. It is significantly important to find a substitute for conjunctival epithelial cells. This study was to explore the possibility of progenitor cell-derived epidermal sheets on denuded amniotic membrane to reconstruct ocular surface of conjunctiva damaged monkeys. We isolated epidermal progenitor cells of rhesus monkeys by type IV collagen adhesion, and then expanded progenitor cell-derived epidermal sheets on denuded amniotic membrane ex vivo. At 3 weeks after the conjunctiva injury, the damaged ocular surface of four monkeys was surgically reconstructed by transplanting the autologous cultivated epidermal progenitor cells. At 2 weeks after surgery, transplants were removed and examined with Hematoxylin-eosin staining, Periodic acid Schiff staining, immunofluorescent staining, scanning and transmission electron microscopy. Histological examination of transplanted sheets revealed that the cell sheets were healthy alive, adhered well to the denuded amniotic membrane, and had several layers of epithelial cells. Electron microscopy showed that the epithelial cells were very similar in appearance to those of normal conjunctival epithelium, even without goblet cell detected. Epithelial cells of transplants had numerous desmosomal junctions and were attached to the amniotic membrane with hemidesmosomes. Immunohistochemistry confirmed the presence of the conjunctival specific markers, mucin 4 and keratin 4, in the transplanted epidermal progenitor cells. In conclusion, our present study successfully reconstructed conjunctiva with autologous transplantation of progenitor cell-derived epidermal sheets on denuded AM in conjunctival damaged monkeys, which is the first step toward assessing the use of autologous transplantation of progenitor cells of nonocular surface origin. Epidermal progenitor cells could be provided as a new substitute for conjunctival epithelial cells to overcome the problems of autologous conjunctiva shortage.
严重的眼表疾病是临床医生目前面临的一些最具挑战性的问题。传统的治疗方法通常效果不佳，这些疾病的长期眼部后果是毁灭性的。找到结膜上皮细胞的替代品非常重要。本研究旨在探索在去上皮化羊膜上培养的表皮祖细胞衍生的表皮片重建猴眼结膜受损的眼表的可能性。我们通过 IV 型胶原黏附分离恒河猴表皮祖细胞，然后在去上皮化羊膜上体外扩增祖细胞衍生的表皮片。在猴眼结膜损伤后 3 周，通过移植自体培养的表皮祖细胞对 4 只猴的受损眼表进行手术重建。术后 2 周，取下移植物，进行苏木精-伊红染色、过碘酸雪夫染色、免疫荧光染色、扫描电镜和透射电镜检查。移植片的组织学检查显示，细胞片健康存活，与去上皮化羊膜紧密黏附，有多层上皮细胞。电镜下观察，上皮细胞与正常结膜上皮细胞的外观非常相似，甚至没有检测到杯状细胞。移植片的上皮细胞有许多桥粒连接，并且通过半桥粒附着在羊膜上。免疫组织化学证实，移植的表皮祖细胞中存在结膜特异性标志物 MUC4 和角蛋白 4。总之，我们的研究成功地在猴眼结膜损伤的去上皮化 AM 上利用自体移植的表皮祖细胞衍生的表皮片重建了结膜，这是评估非眼表来源的自体祖细胞移植的第一步。表皮祖细胞可以作为结膜上皮细胞的新替代品，以克服自体结膜短缺的问题。
