Abstract
Purpose::
Retinal stem cells (RSCs)/progenitors are critical for the development of therapeutic approaches for degenerative retinal diseases. Lentivirus-mediated gene transfer is an important strategy for RSC ex vivo modulation. This study is to investigate long-term gene expression mediated by lentiviral vector in RSCs.
Methods::
RSCs were isolated from mouse retina at postnatal day 1 and cultured in serum-free culture medium supplemented with EGF and FGF. Retinal spheres were transduced with GFP-expressing feline immunodeficiency virus (FIV). GFP expression was analyzed by flow cytometry and fluorescent microscope. RSCs were differentiated in mitogen-free culture medium supplemented with 5% FBS. Expression of cell-specific markers was analyzed by immunocytochemistry in both undifferentiated and differentiated RSCs. Differentiated postmitotic cells were revealed by BrdU incorporation assay.
Results::
Stem cell marker nestin was detected in the cultured retinal spheres. FIV transduced more than 97% of retinal spheres with sustainable GFP expression for at least two months in culture. Upon differentiation, cell-specific markers, including MAP-2, opsin and GFAP, were detected in FIV-transduced or control RSCs at similar levels, suggesting that FIV transduction did not affect cell differentiation. However, GFP expression substantially decreased in BrdU-negative differentiated cells, suggesting the downregulation of the transgene expression in differentiated postmitotic cells.
Conclusions::
FIV-mediated gene transfer induces sustainable gene expression in undifferentiated RSCs. However, the transgene expression is down-regulated in differentiated RSCs.
Keywords: regeneration • gene transfer/gene therapy • differentiation