Conjunctival Reconstruction with Progenitor Cell-Derived Autologous Epidermal Sheets in Rhesus Monkey

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.严重眼表疾病是当前临床医师面临的极具挑战性的难题之一。常规治疗方案通常难以取得满意疗效，此类病症带来的长期眼部并发症后果极为严重。寻找结膜上皮细胞的替代材料具有至关重要的临床意义。本研究旨在探索利用祖细胞来源的表皮片层构建于脱上皮羊膜（denuded amniotic membrane）之上，以修复结膜损伤恒河猴眼表的可行性。我们通过IV型胶原黏附法分离恒河猴的表皮祖细胞，随后在体外扩增细胞并制备为表皮片层，将其接种于脱上皮羊膜之上。在结膜损伤造模3周后，我们通过手术移植自体培养的表皮祖细胞片层，对4只恒河猴受损的眼表进行重建修复。术后2周，取出移植片并通过苏木精-伊红（Hematoxylin-eosin）染色、过碘酸-希夫（Periodic acid Schiff）染色、免疫荧光（immunofluorescent staining）染色，以及扫描电子显微镜、透射电子显微镜进行检测分析。对移植片的组织学检查结果显示，细胞片层存活状态良好，与脱上皮羊膜贴合紧密，且形成了多层上皮细胞结构。电子显微镜观察表明，移植的上皮细胞形态与正常结膜上皮细胞高度相似，即便未检测到杯状细胞（goblet cell）。移植的上皮细胞存在大量桥粒连接（desmosomal junctions），并通过半桥粒（hemidesmosomes）附着于羊膜基质。免疫组织化学检测证实，移植的表皮祖细胞表达结膜特异性标志物黏蛋白4（mucin 4）与角蛋白4（keratin 4）。综上，本研究成功在结膜损伤的恒河猴模型中，通过将祖细胞来源的表皮片层移植于脱上皮羊膜之上完成了结膜结构的重建，这一成果为评估非眼表来源的祖细胞移植应用迈出了关键的第一步。表皮祖细胞可作为结膜上皮细胞的新型替代材料，解决自体结膜组织来源不足的临床难题。