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Victoria Tovell, Britta Nommiste, Amanda-Jayne Francis Carr, Lyndon daCruz, Peter J Coffey; Transcriptome and morphological analysis of endothelial cells co-cultured with retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3259.
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© ARVO (1962-2015); The Authors (2016-present)
A tissue engineered choroid will, together with the retinal pigment epithelial (RPE) cells, provide a useful tool to help us understand RPE/choroid disease mechanisms, and will ultimately help develop a functionally and structurally accurate choroid for transplantation. Here we investigate changes in the transcriptome and morphology of endothelial cells (EC), when co-cultured with RPE cells in a 3D culture system, to look for evidence of a choroid specific phenotype.
RPE cells were derived from iPSC by spontaneous differentiation and maintained in X-VIVO media. Endothelial cells derived from iPSC (iEC, Cellular Dynamics International) were maintained in VEGF media according to manufacturer’s instructions. Polyester membranes of a transwell insert (6.5 mm diameter, 0.4 um pore size) were coated on both sides with matrigel for 1 hour at 37oC. RPE and ECs were seeded on opposite sides of the transwell membranes at 50,000 cells/cm2 and 10,000 cells/cm2 respectively. Cultures were maintained in cell specific media (RPE in X-VIVO and ECs in VEGF media) for 1, 3 and 6 weeks before RNA was isolated separately from each cell type. RNA seq was used to analyse the transcriptomes of ECs in co-culture compared with control. Confocal microscopy was used to capture z-stack images of the 3D co-culture in order to assess morphological changes.
RPE was successfully derived from iPSC and displayed classic RPE cell morphology, pigmentation and expression of RPE specific markers including PMEL17. Transcriptional profiling suggests that co-culture of RPE cells with endothelial cells results in differential gene expression, when compared to RPE or iEC cells cultured alone.
Preliminary studies suggest that co-culturing ECs with RPE may be useful when driving iPSC-ECs into a choroid specific phenotype.
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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