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Clarisse Fligor, Sarah Ohlemacher, Donald J Zack, Jason S Meyer; Axonal Outgrowth and Regeneration From Human Pluripotent Stem Cell-Derived Retinal Ganglion Cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):6077. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Human pluripotent stem cells (hPSCs) possess the unique ability to differentiate into any cell type in the body, thereby allowing them to serve as a platform for the study of retinal differentiation and regeneration as well as the development of translational therapies. hPSCs are especially relevant for studies of retinal degenerative diseases and injury, where the axons of retinal ganglion cells (RGCs) are lost. In this role, hPSCs can serve as an in vitro model for axonal outgrowth and regeneration.
hPSCs were differentiated to a retinal lineage following established protocols and RGCs were identified by expression of BRN3, as well as their expression of a variety of other RGC-associated features. Presumptive RGCs could also be identified with an mCherry reporter linked to BRN3. The ability of these cells to extend neurites as an in vitro model for axonal outgrowth and regeneration was examined by systematically dissociating and plating cell aggregates. After 24 hours, cells were fixed and analyzed by immunocytochemistry to quantify the extent of neurite growth. Factors influencing this process, including substrates, culture medium, or candidate chemoattractant compounds were also tested and their effects on neurite outgrowth was compared to control experiments.
Within 40 days of differentiation, abundant RGC differentiation was observed from hPSCs, tightly correlated with the expression of the mCherry reporter. Immunocytochemistry analysis confirmed that BRN3-expressing presumptive RGCs also expressed many RGC-associated features. mCherry-expressing RGCs began to extended neurites within hours of plating, with noticeable increase in length by 24 hours. To examine the factors influencing neurite outgrowth, the effects of varying substrates as well as culture medium formulations was examined. With the goal of eventually being able to direct neurite outgrowth, the effects of candidate chemoattractant compounds was also tested.
Overall, these results demonstrate the utility of hPSC-derived RGCs for studies of axonal outgrowth and regeneration. RGCs were capable of rapid neurite outgrowth, with these neurites extending for significant distances. These results provide a novel in vitro assay for testing the ability of RGCs to extend axons and may assist in the development of personalized transplant therapies for optic neuropathies.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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