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Philip Wagstaff, Anneloor ten Asbroek, Valeria Lo Faro, Shazia Micheal, Nomdo M Jansonius, Arthur A Bergen; Utilising a 3D Matrigel stem cell culture leads to a significantly shorter time-span for generating retinal ganglion cells.. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3747.
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© ARVO (1962-2015); The Authors (2016-present)
Glaucoma is a debilitating group of diseases that cause an insidious and irreversible loss of retinal ganglion cells (RGCs) in affected eyes. Current treatments focus on lowering the intraocular pressure to slow down further damage of remaining retinal ganglion cells, but do not take into account those that have already been lost. Stem cell research is revolutionising glaucoma treatment, with the ability to differentiate RGCs from stem cells paving the way to new in vitro models and future cell replacement therapies. The current method of generating RGCs includes a lengthy floating 3D culture to produce embryoid bodies. This study aimed to reduce this 3D step considerably by way of a more robust protocol that significantly reduces the time to produce ganglion cells in vitro.
Induced pluripotent stem cells (iPSCs) were encased in 3D Matrigel drops for 5 days before being dissociated and replated in a 2D culture. Known RGC promoting components were added to the medium, culturing cells long term. qPCR was completed on samples taken every 2 days to detect RGC specific marker expression, and samples were also stained for visual representation. In parallel, fibroblasts were taken from clinically well characterised glaucoma patients and reprogrammed into iPSCs.
We found that embryoid bodies of similar size could be grown in 5 days while encased in Matrigel, compared to over 25 days with the different floating culture methods used by other groups. Both embryonic and induced pluripotent stem cells behaved in a similar fashion throughout the differentiation protocol, and the expression of retinal lineage markers mirrored the order found in vivo, including RGC specific markers BRN3, ISL-1, RBPMS and ATOH7. Staining the whole 3D drop showed complex embryoid body structure with early developmental marker expression. Cells from patients with known glaucoma mutations, including CYP1B1, were successfully reprogrammed to an induced pluripotent state.
Our results present an alternative protocol for differentiating retinal ganglion cells in an efficient and robust manner. The sequential expression of known markers, emulating the similar in vivo route, give a good indication of the general quality of RGCs produced. Overall, this is a promising beginning for future cell replacement therapies.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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