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Rebecca J Fawcett, Yan Guo, Candace Kerr, Steven L Bernstein; Cultivation of mouse neural progenitor cells from the optic nerve. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3328.
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© ARVO (1962-2015); The Authors (2016-present)
We previously reported expression of nestin and SOX-2 positive cells in the mouse optic nerve (ON)(Guo et al, ARVO 2015). These proteins are known to be localized in the CNS in neural progenitor cells found in the subventricular zone (SVZ) and in the hippocampus. We wanted to culture nestin/Sox 2(+) cells from the ON, and determine their growth potential and stem cell marker profile, in order to gain insight into the unique growth conditions that may aid in both self-renewal and ON repair
Optic nerve tissue was dissected, pooled and dissociated from 17-day old mice (n=8 mice/group), using collagenase. Cells were resuspended in supplemented serum-free neurobasal medium. Cell colonies were derived from the resuspended cells, and allowed to expand for various immunohistochemical, and molecular analyses. Cells were fixed in situ with paraformaldehyde and immunostained using nestin and SOX-2 antibodies, and visualized by confocal microscopic analysis. qPCR on first strand cDNA was performed to quantify levels of stem- and neural cell markers, and compared against freshly isolated ON and retina.
Immunostaining of cultured cells showed positive co-expression for both nestin and SOX-2. In wells supplemented without serum in the presence of platelet-derived growth factor (PDGF), many, but not all ON-derived cells differentiated into olig1+, O4+ oligodendrocyte progenitors. RT-qPCR quantification of cDNA derived from mouse ON-derived cells revealed higher levels of nestin and Sox2, compared with ON and retinal tissue.
Cells derived from the mouse ON exhibit the capacity for self-renewal, similar to those seen for progenitors from other neural sources. These cells undergo successful continuous proliferation after 6 months. Our findings suggest a path for additional studies into ON-derived cells that may be useful in defining the unique conditions needed to obtain cells capable for studies into ON repair and regeneration.
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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