Purchase this article with an account.
Jeevan Srinivas Rajkumar, Michael Maddison, Deepti Singh, PIERRE COLOMBE, Myron Spector, Michael J Young; Enrichment of a Retinal Ganglion Cell Population using a Novel Microfluidic Sorting Strategy. Invest. Ophthalmol. Vis. Sci. 2020;61(7):2521.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Cell therapy provides a novel approach to restoration of vision for those who suffer from glaucoma other degenerative diseaes that impact the optic nerve (e. g. neurofibromatosis type1. Specifically, retinal ganglion cells (RGCs) transplantation may someday restore visual function for those with damaged optic nerves in these conditions. This study evaluated different cell surface markers that may enable one to successfully sort and purify RGCs from mixed neuronal populations for transplantation studies.
Mouse embryonic stem cells were cultured through established media/conditions to form optic cups. They were then sorted with a Miltenyi Tyto cell sorter using Thy1.2+ (labels RGCs) L1-CAM+ (labels RGC axons) CD-73- (labels photoreceptors) to optimize enrichment and prevent photoreceptor contamination. To verify RGC markers and the efficacy of negative sorts, post-sort populations were characterized with BRN3a, Thy1.1, Thy1.2, NeUN (RGC expression markers) and Arrestin, CRX, Recoverin, Ki67, and Oct3 (photoreceptor and other ell type markers). A live-dead assay was also performed.
Thy1.2+L1-CAM+CD-73- as a RGC marker set sorts with ~95% purity. Importantly, the this method led to high viability (~90%), due to the gentle microfluidic sorting technique of the Tyto. Characterization shows that all RGC markers are significantly prominent in positive sort populations whereas all photoreceptor markers are notably lower than control. Ki67 confirms that these cells can be proliferated to maintain population growth.
The study was successful in optimizing a protocol to purify an enriched population of retinal ganglion cells. It verifies the previously-unknown phenotype of Thy1.2+L1-CAM+CD-73- as an effective strategy to isolate cells that are both pure and viable for transplantation. Future studies will study the survival and engraftment of these cells after implantation in the retina.
This is a 2020 ARVO Annual Meeting abstract.
This PDF is available to Subscribers Only