Regeneration of the corneal epithelium could be severely impaired in patients suffering from limbal stem cell deficiency(LSCD).The purpose of this study was to evaluate the restoration of the corneal epithelium by grafting onto denuded corneas human limbal cells cultured on human amniotic membrane.

Retinal pigmented epithelium (RPE) cells was obtained from neonatal cadaver globes, and cultured in DMEM:F12 supplemented with 10% FBS. In definite passages, cells were trypsinized and co-cultured with 30% AF (from normal fetuses with gestational ages of about 14-16 weeks) on scaffolds of amniotic membrane. RPE markers (RPE65 & cytokeratin) was assessed by immunocytochemical (ICC) analysis. Retinal progenitor markers' (PAX6, CHX10) expression was evaluated both through ICC analysis and real-time polymerase chain reaction (RT-PCR).

RPE cell culture was established on AM surface successfully (Fig 1. A & B). ICC analysis showed that cells cultured on AM in the presence of 30% AF were positive for RPE65 (Fig 1. C & D) and cytokeratin (not shown), confirming the RPE identity of the cultivated cells. PAX6 and CHX10 were also detected in the cell sheets immunocytochemically. mRNA expression of PAX6 and CHX10 displayed a significant increase in 30% AF treated cells cultured on AM compared to those cultivated in the presence of 10% FBS on the plate surface (37 & 588 fold respectively) (Fig 1. E).

There is a huge bulk of study improving the impressive effect of AM and AF on differentiation or of the cultured cells (4, 5). Based on the presenting results, concurrent utilization of AM (as scaffold) and AF (as supplementary medium) can produce significant increase in progenitor markers in RPE cells, compared with FBS treated RPEs. Our results indicate the potential of AM-AF combination to promote retinal progenitor cells which is promising in regenerative medicine.