P-147: Neural Progenitor Cells Arising from Amniotic Fluid (AF) Treated Retinal Pigment Epithelium (RPE) Cell Cultures


Sanie-Jahromi F *, Soheili Z , Ahmadieh H , Davari M , Ghaderi Sh , Rezaie Kanavi M , Samiei Sh ,

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Objective: Seeking for retinal-neural progenitor cells as a prospective therapeutic substrate for eye diseases, RPE cell cultures were established and treated with amniotic fluid (AF). AF is an organic pure composite having tremendous proliferative impacts on multipotent embryonic cells. AF effects on the cultured RPE cells were evaluated utilizing several methods. Materials and Methods: RPE cells harvested from neonatal cadaver globes, obtained from Central Eye Bank of Iran, were cultured in Dulbecco's Modified Eagle Medium (DMEM):F12 supplemented with 10% FBS. In definite passages, cells were trypsinized and co-cultured with 30% AF (obtained from normal fetuses with gestational ages of about ≤ 12 weeks). AF inductive growth effect on RPE cells from different passages was assessed employing ELISA cell proliferation and cell death kit according to the supplier instruction manual (Roche, Germany). Retinal progenitor markers (Pax6 & Chx10) expression was assessed and enumerated through immunocytochemical analysis of RPE cells from passage 6, according to Santa Cruz protocol. Confirming the previous data, RNA extraction (QIAGEN, Germany), cDNA synthesis and real time polymerase chain reaction (RT-PCR) (Roche, Germany) were also performed. Results: ELISA assay represented a salient increase in RPE cells' growth rate cultivated in 30% AF, compared with those grown in the absence of AF. No meaningful disparity in proliferation rate was discerned between treated and control cultures. Immunocytochemical analysis exhibited nuclear localization of progenitor markers in a ratio of 33% and 27% for chx10 and pax6 respectively. This indicated a 3 fold raise in AF treated cultures compared to control cultures, Real-Time PCR data was concurring with the foregoing results approving the AF capacity of promoting progenitor cell propagation. Conclusion: The presented data imply the phenomenal influence of AF on RPE cells’ culture, indicating its qualified fitness for FBS replacement in the medium and its potential to induce RPE cells proceeding toward progenitor cells. These progenitor cells can be a convenient source for cell replacement therapy in retinal diseases