Session Title: New drug treatment and technology
Session Date/Time: Friday 27/09/2013 | 14:30-16:00
Paper Time: 15:18
Venue: Hall 3 (Level 0)
First Author: Z.Soheili IRAN
Co Author(s): E. Darbari H. Rezanejad S. Samiei H. Ahmadieh
Retinal pigment epithelium cells (RPE) form a pigmented layer in the inner surface of retina. RPE cells can be reprogrammed to produce progenitor cells, so they can be used as potential source for cell therapy purposes. SOX2 is a transcription factor and an important marker for neural progenitors and stem cells in CNS in vertebrate. By expression of SOX2, neural progenitor/stem cells characteristics will be retained. The purpose of this study was to dedifferentiate RPE cells to neural progenitor cells by SOX2 over expression.
National institute of Genetic Engineering and Biotechnology
RPE cells were isolated from human eye globes and cultured in Dulbecco's Modified Eagle's Medium (DMEM) /Ham’s F-12 supplemented with 20% fetal bovine serum (FBS). They were subcultured in DMEM/Ham’s F-12 with 10% FBS in subsequent passages. Coding region of SOX2 gene was synthesized and cloned into PCR2.1 vector, then by BamHI and XhoI restriction enzymes, SOX2 gene was digested and subcloned into pLEX- MCS Lentiviral vector which contained puromycin resistant gene as selectable marker. Accuracy of recombinant construct was approved by subsequent nucleic acid sequencing. Recombinant construct with helper vectors were transfected to HEK293T using calcium phosphate method and virus-containing media was collected from transfected HEK293T cultures. Subsequently, RPE cells were infected and after 96 hours were treated with 0.7 µg/mL of puromycin. After 72 hours, RPE cells showed resistant against antibiotic. ICC and Real Time PCR methods evaluated the rate of expression of neural progenitor cells’ markers. Nestin, PAX6, CHX10 were used as neural progenitor cells’ markers and Thy1 and Rho were used as retinal neural cell’s markers.
Real Time PCR and ICC showed strong expression in SOX2, PAX6, CHX10 and Nestin as neural progenitor markers. Interestingly the expression of Thy1 and Rho was also determined. These data showed reprogramming and transdifferentiation were occurred in treated RPE cells respect to control.
Successful infection of RPE cells by desired vectors made a promise for following SOX2 gene over expression and regeneration of retinal progenitor/stem cells in cultures conjointly with retinal ganglion cells and photoreceptors.