Session Title: New Drug Treatment and Technology II
Session Date/Time: Sunday 20/09/2015 | 09:00-10:30
Paper Time: 09:24
First Author: : Z.Soheili IRAN, ISLAMIC REPUBLIC OF
Co Author(s): : R. Ezzati E. Ranaei Pirmardan S. Samiei H. Ahmadieh
PURPOSE:Retinal degeneration causes blindness. One potential therapy is cell replacement. Since the human retina lacks regeneration capacity much attention has been directed towards searching for cells that can differentiate into retinal neurons. In this study we investigated the role of SOX2 over-expression in human retinal pigment epithelial (RPE) cells culture fate.
In this study we investigated the role of SOX2 over-expression in human retinal pigment epithelial (RPE) cells culture fate.
coding sequence of human SOX2 gene was cloned in pAAV-MCS vector. Green fluorescent protein (GFP) cloned in downstream of SOX2 gene as a reporter gene for determining gene transfection efficiency. The aforementioned vector in association with two other essential vectors of AAV helper free system was transfected to HEK293T cells and propagated virus titer was determined by flowcytometry. Human newborn RPE cells were cultured and infected by recombinant virus. Real time PCR and immunocytochemistry (ICC) was performed to analyze the effect of SOX2 over-expression in RPE cells’ culture fate.
SOX2 and GFP cloning were confirmed by PCR, restriction digestion and sequencing. Flow cytometry results showed the presence of 5×106 particles in each ml of prepared virus stock. Human newborn RPE cells were infected by recombinant virus, with multiplicity of infection (MOI) equal to 10. For treated and control cultures, mRNA extraction and real time PCR was done and the results showed 80 fold increases for SOX2 in infected RPE cells. To investigate the effects of SOX2 over-expression in infected RPE cells’ culture, real time PCR was performed for retinal primitive cells’ markers including PAX6 and CHX10 and neural progenitor cells’ marker; nestin . Result showed nestin over-expression in treated cells. Nestin positive cells were detected by ICC and real time PCR data was confirmed.
Previous studies had revealed that expression of SOX2 inhibited neuronal differentiation and induced maintenance of progenitor cells’ trait in primitive cells. On the other hand inhibition of SOX2 signaling was associated with loss of progenitor cells’ markers and the onset of early neuronal differentiation markers in treated cells. Our study showed that SOX2 over-expression induced a meaningful increase in nestin expression in treated cells. Increased nestin expression, as neural progenitor cells’ marker, suggests that expression SOX2 could stimulate RPE cells to dedifferentiate towards neural progenitor cells.