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Yang Liu, Gaurang C Patel, Weiming Mao, Abbot F Clark; Establishment of conditionally immortalized mouse optic nerve astrocyte line. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2548. doi: https://doi.org/.
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Optic nerve astrocytes play a major role in axonal degeneration and regeneration. Astrocyte lines are an important tool to elucidate the cellular mechanisms responsible. In this study, we established a conditionally immortalized mouse optic nerve astrocyte line.
Astrocytes were cultured from explants obtained from postnatal day 5-7 H-2kb-tsA58 transgenic mouse optic nerves. Cells were cultured in defined astrocyte culture medium under permissive (33°C) or non-permissive (38.5°C) temperature with or without the presence of interferon-γ (IFN-γ). Astrocytes were characterized by immunocytochemistry staining using antibodies against glial fibrillary acidic protein (GFAP) and neural cell adhesion molecule (NCAM). Cell proliferation rates were determined by cell growth curves and percentage of Ki67 positive cells. Karyotyping was performed to validate the mouse origin of established cell line. Conditional immortalization was assessed by western blot-determined expression levels of SV40 large T antigen (TAg), p53, GFAP and NCAM in non-permissive culture conditions. In addition, phagocytic activity of immortalized cells was determined by flow cytometry-based pHrodo florescence analysis.
After 5 days in culture, cells migrated out from optic nerve explants. Immunocytochemistry staining showed that migrating cells expressed astrocyte makers, GFAP and NCAM. In permissive conditions, astrocytes had increased expression levels of TAg and p53, a greater cell proliferation rate as well as a higher percentage of Ki67 positive cells (n=3, p<0.05) compared to cells cultured in non-permissive conditions. One cell line (named ImB1ON) was further maintained through 60 generations. Karyotyping showed that ImB1ON was of mouse origin. Flow cytometry-based pHrodo florescence analysis demonstrated phagocytic activity of ImB1ON cells. Non-permissive culture conditions decreased expression of p53 and TAg in ImB1ON, and increased expression of GFAP and NCAM.
A conditionally immortalized mouse optic nerve astrocyte line was established. This cell line provides an important tool to study astrocyte biological processes.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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