Abstract Form

Title: In Vivo Evaluation of PAX6 Overexpression and NMDA cytotoxicity to Stimulate Proliferation in the Mouse Retina
Author(s): Ehsan Ranaei Pirmardan, Zahra-Soheila Soheili, Shahram Samiei, Hamid Ahmadieh, Seyed Javad Mowla, Narsis Daftarian, Marzieh Naseri
Presentation Type: Oral
Subject: Retina and Retinal Cell Biology
Presenting Author:
Name: Ehsan Ranaei Pirmardan
Affiliation :(optional) Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
E mail: ranaei_ehsan@yahoo.com
Phone: 44456653
Mobile: 09151075236
Abstract (Max 200 words)
Purpose: In eye retina, due to the lack of regeneration systems and self-renewable cells, degenerative diseases often lead to a visual impairment. One of the aims of the regenerative medicine is conversion of a cell type into another. Although trans-differentiation approaches were previously stablished and several reports are in hand, successful reprogramming of adult cells by ancestral transcription factors was a milestone in cell biology. The Pax6 transcription factor has a key role in eye development and retinal development. The main aim of this project was to investigate in vivo Pax6 overexpression, mediated by adeno-associated virus (AAV) and induction of retinal ganglion cell death to induce retro-differentiation of retinal cells and commit them to re-enter into the cell cycle.
Methods: Recombinant AAV virus harboring PAX6 cDNA and reporter gene, EGFP, was produced, purified and concentrated with heparin sulfate and amicon columns. Virus titration was performed with flow cytometry and qPCR. Viruses were injected into untreated and model mouse eyes. Induction of acute retinal ganglion cell death and generation of mouse experimental model was performed by N-methyl D-aspartic acid (NMDA) injection. All experiments including virus injection, histology and expression analyses with IHC and retinal flatmount were done.
Results: After a plenty of time consuming technical setup, we achieved to a high transgene expression in mouse retina. We did not observe any changes in Ki67 expression, cell cycle marker, following PAX6 overexpression in both of the model mice and its controls. However, we detected expression of progenitor cell marker, Sox2, in photoreceptors layer in treated animals.
Conclusion: We produced in vivo grade rAAV viruses, performed intraocular injection of viral particles and did subsequent analyses for the first time in Iran. Despite to the distinct reports on Pax6 functions in development and cancers, it assumed that PAX6 overexpression could not reprogram retinal cells to progenitor cells. Nevertheless we found that photoreceptor cells expressed Sox2, an important progenitor cells’ marker. This is a very rare event that promises high cellular plasticity of retinal cells and a vivid future for retinal regenerative medicine.