Establishment of a conditionally immortalized mouse optic nerve astrocyte line

Yang Liu, Gaurang C. Patel, Weiming Mao, Abbot F. Clark

Research output: Contribution to journalArticle

Abstract

Optic nerve astrocytes play a major role in axonal degeneration and regeneration. Astrocyte lines are an important tool to elucidate the responsible cellular mechanisms. In this study, we established a conditionally immortalized mouse optic nerve astrocyte line. Astrocytes were cultured from explants derived from postnatal day 4–5 H-2k b -tsA58 transgenic mouse optic nerves. Cells were cultured in defined astrocyte culture medium under permissive (33 °C) or non-permissive (38.5 °C) temperatures with or without interferon-ɤ (IFN-ɤ). 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 fluorescence 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, exhibited 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 (ImB1ON) was further maintained through 60 generations. Karyotyping showed that ImB1ON was of mouse origin. Flow cytometry-based pHrodo fluorescence analysis demonstrated phagocytic activity of ImB1ON cells. Quantitative PCR showed mRNA expression of trophic factors. Non-permissive culture conditions decreased expression of TAg and p53 in ImB1ON, and increased the expression of NCAM. A conditionally immortalized mouse optic nerve astrocyte line was established. This cell line provides an important tool to study astrocyte biological processes.

Original languageEnglish
Pages (from-to)188-195
Number of pages8
JournalExperimental eye research
Volume176
DOIs
StatePublished - Nov 2018

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Optic Nerve
Astrocytes
Neural Cell Adhesion Molecules
Viral Tumor Antigens
Glial Fibrillary Acidic Protein
Karyotyping
Cell Line
Cultured Cells
Flow Cytometry
Fluorescence
Immunohistochemistry
Cell Proliferation
Staining and Labeling
Polyomavirus Transforming Antigens
Biological Phenomena
Interferons
Transgenic Mice
Culture Media
Regeneration
Cell Culture Techniques

Keywords

  • Cell line
  • Conditional immortalization
  • Optic nerve astrocyte

Cite this

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title = "Establishment of a conditionally immortalized mouse optic nerve astrocyte line",
abstract = "Optic nerve astrocytes play a major role in axonal degeneration and regeneration. Astrocyte lines are an important tool to elucidate the responsible cellular mechanisms. In this study, we established a conditionally immortalized mouse optic nerve astrocyte line. Astrocytes were cultured from explants derived from postnatal day 4–5 H-2k b -tsA58 transgenic mouse optic nerves. Cells were cultured in defined astrocyte culture medium under permissive (33 °C) or non-permissive (38.5 °C) temperatures with or without interferon-ɤ (IFN-ɤ). 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 fluorescence 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, exhibited 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 (ImB1ON) was further maintained through 60 generations. Karyotyping showed that ImB1ON was of mouse origin. Flow cytometry-based pHrodo fluorescence analysis demonstrated phagocytic activity of ImB1ON cells. Quantitative PCR showed mRNA expression of trophic factors. Non-permissive culture conditions decreased expression of TAg and p53 in ImB1ON, and increased the expression of NCAM. A conditionally immortalized mouse optic nerve astrocyte line was established. This cell line provides an important tool to study astrocyte biological processes.",
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Establishment of a conditionally immortalized mouse optic nerve astrocyte line. / Liu, Yang; Patel, Gaurang C.; Mao, Weiming; Clark, Abbot F.

In: Experimental eye research, Vol. 176, 11.2018, p. 188-195.

Research output: Contribution to journalArticle

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AB - Optic nerve astrocytes play a major role in axonal degeneration and regeneration. Astrocyte lines are an important tool to elucidate the responsible cellular mechanisms. In this study, we established a conditionally immortalized mouse optic nerve astrocyte line. Astrocytes were cultured from explants derived from postnatal day 4–5 H-2k b -tsA58 transgenic mouse optic nerves. Cells were cultured in defined astrocyte culture medium under permissive (33 °C) or non-permissive (38.5 °C) temperatures with or without interferon-ɤ (IFN-ɤ). 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 fluorescence 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, exhibited 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 (ImB1ON) was further maintained through 60 generations. Karyotyping showed that ImB1ON was of mouse origin. Flow cytometry-based pHrodo fluorescence analysis demonstrated phagocytic activity of ImB1ON cells. Quantitative PCR showed mRNA expression of trophic factors. Non-permissive culture conditions decreased expression of TAg and p53 in ImB1ON, and increased the expression of NCAM. A conditionally immortalized mouse optic nerve astrocyte line was established. This cell line provides an important tool to study astrocyte biological processes.

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