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Marina Zalis, Sebastian Johansson, Ulrica Englund Johansson, ; The combination of neurotrophic factors and laminin substantially promote retinal ganglion cell axon outgrowth after dissociation. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2255.
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© ARVO (1962-2015); The Authors (2016-present)
Laminin, present in the optic nerve, promotes regrowth of injured retinal ganglion cell (RGC) axons in lower species. However, a permissive substrate alone is not enough to promote axonal regeneration in adult mammals. Hence, we wanted to explore the regenerative potential of dissociated retinal cells on a laminin functionalized substrate and a RGC-specific enriched medium as a first step towards cell therapy in retinal degenerative disease.
Isolated post-natal day 4 mouse retinas were dissociated to single cell suspension. Cells were cultured on 4-well-chamber slides coated with Poly-L-Lysine (PLL) or PLL + Laminin for 7 days in vitro (DIV). Either basic neuronal medium (DMEM-F12, 2% B27 supplement) or the enriched so called Full-SATO (Neurobasal, CNTF, BDNF, Forskolin, Insulin) medium was used. Immunohistochemistry using cell specific markers were used to identify RGCs (i.e. NeuN, β-Tubulin III and RBPMS) and glial cells (GFAP).
Repeated studies comparing the effect of basic neuronal medium and Full-SATO medium on the overall survival of retinal cells cultured on PLL coated slides, revealed no obvious difference up to 7 DIV. Cells cultured with basic neuronal medium on PLL coated slides mainly appeared as single cells, with round cell bodies and short processes, except for a modest GFAP+ cell population that displayed a polygonal morphology. In contrast, Full-SATO medium induced both a significant increase in numbers of GFAP+ and RBPMS+ cells, and more complex cellular profiles. Addition of laminin further increased numbers of GFAP+ and RBPMS+ cells using Full-SATO medium, compared to PLL counterparts. These cells formed clusters, displayed extensive neurite outgrowth (NeuN+, β-Tubulin III+, even GFAP+) and highly complex cell morphologies.<br /> In all culture conditions, cells co-expressing GFAP and RBPMS were found, suggesting a progenitor state.
We have shown that the trauma of axotomy due to dissociation can be overcome in vitro using a laminin coated substrate, and an enriched RGC-specific medium, which resulted in substantial axonal outgrowth. Our finding is an important step for understanding the environmental conditions needed in successful cell-based restorative therapies for retinal degenerations.
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