Effects of amniotic membrane on proliferation and differentiation of human retinal pigment epithelial cell

Background: Human retinal pigment epithelial (RPE) cell transplantation treating retinal degenerative diseases is a researching topic, and the source of human RPE cells is a key problem. Many biological carriers can be used for the preparation of RPE cell layer. However, some advantages, such as cytotoxicity, lack of stability and immunologic reaction etc. are still existed. To study an ideal biological carrier is very important. Objective: This experimental was to determine the effects of amniotic membrane on the proliferation and differentiation of human RPE cells and the possibility as a scaffold for RPE cell transplantation. Methods: ARPE-19 cell line cells were cultured and passaged in DMEM/F12 medium with 10% fetal bovine serum, and 8-12 generation of cells were used. The cells were divided into two groups. One group of cells were incubated on the denuded amniotic membrane, and the other group of cells were cultured in the medium (control group). MTT was performed to detect the A₄₉₂ value of RPE cells for the evaluation of cell proliferation ability 24, 48, 72, 96 hours after culture. Cell morphology was compared by histopathological examination 3 weeks after culture. The mRNA expression of pigment epithelium-derived factor (PEDF), N-cadherin, β-catenin and cell connection related proteins in the cells of both groups were assayed using reverse transcription polymerase chain reaction (RT-PCR). Ultrastructure of the cells was observed under the transmission and scan electronic microscope 3 weeks after culture. Results: The number of ARPE-19 cells cultured on denuded amniotic membrane was decreased significantly in comparison with the normal culture plate (F=41.760, P=0.000). Histopatholy also showed that the cell density on amniotic membrane was lower than of normal cells on plate surface. Moreover, the expression level of claudin 1 mRNA, N-cadherin mRNA and PEDF mRNA were significantly up-regulated in denuded amniotic membrane group in comparison with control group (t=15.828, P=0.000; t=6.839, P=0.002; t=14.667, P=0.000), but the expression of Connexin 43 mRNA was down-regulated in denuded amniotic membrane group compared with control group(t=3.358, P=0.024). Ultra-structural examination revealed that ARPE-19 cells cultured on amniotic membrane exhibited a polygonal epithelial phenotype with cilium on the apical side, however, the cells cultured on normal culture plate displayed fusiform shape and uneven thickness. Conclusions: Amniotic membrane plays a promoting effect on the differentiation of ARPE-19 cells and a inhibitory effect on the proliferation of ARPE-19 cells, suggesting that amniotic membrane might be an useful scaffold for the preparation of functionally mature RPE cells for clinical transplantation.