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Maciej Daniszewski, Duncan Crombie, Louise Rooney, Alison Conquest, Alex W. Hewitt, Alice Pebay; Refining retinal ganglion cell (RGC) differentiation from human pluripotent stem cells (hPSCs). Invest. Ophthalmol. Vis. Sci. 2018;59(9):3746. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Glaucoma is an optic neuropathy characterized by gradual degeneration of RGCs and their axon leading to irreversible vision loss. The molecular profiling of human RGCs in normal and diseased ocular tissue is hindered by the lack of non-invasive means of obtaining RGCs from living donors. This issue can now be circumvented by the use of hPSCs as a source of RGCs. Three-dimensional (3D) hPSC-derived organoid cultures are thought to better recapitulate normal development than 2-dimensional (2D) adherent cell models. We investigate feasibility of using a 3D model to obtain RGCs for downstream applications.
Human pluripotent stem cells (hPSCs) were differentiated into optic cups following methods developed by Sasai and colleagues and Goureau and colleagues. Organoids were harvested at different time points, dissociated into single cells and sorted using magnetically activated cell sorting (MACS) with THY1 (RGC marker). Recovered cells were subsequently characterised by immunostaining for various cell markers, including RGCs (BRN3B), astrocytes (GFAP) and photoreceptors (CRX). qPCR analysis of gene expression was also performed.
Optic cups were maintained for various time points and subsequently analysed by qPCR to assess cellular populations, revealing gradual increase in expression of genes characteristic for RGCs (THY1, ATOH7), retinal pigment epithelium (RPE65, CRALBP) and photoreceptors (CD73, RECOVERIN, RHO, OPN1SW, OPN1MW). Cells isolated by MACS from optic cups at various time points were plated onto Matrigel and maintained in Pro-B27 medium for 4 days. Immunocytochemistry and qPCR demonstrated the presence of RGCs, as well as carryover of other cell types of the neural retina and progenitor cells in the culture.
MACS of the hPSC-derived optic cups enabled us to isolate RGCs. This will allow us to conduct transcriptomic studies of organoid-derived RGCs. However, further work is also required to scale and simplify differentiation protocols in order to obtain large quantities of cell type of interest at high purity.
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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