July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Transcriptome and morphological analysis of endothelial cells co-cultured with retinal pigment epithelial cells
Author Affiliations & Notes
  • Victoria Tovell
    University College London, London, United Kingdom
  • Britta Nommiste
    University College London, London, United Kingdom
  • Amanda-Jayne Francis Carr
    University College London, London, United Kingdom
  • Lyndon daCruz
    University College London, London, United Kingdom
  • Peter J Coffey
    University College London, London, United Kingdom
  • Footnotes
    Commercial Relationships   Victoria Tovell, None; Britta Nommiste, None; Amanda-Jayne Carr, None; Lyndon daCruz, None; Peter Coffey, None
  • Footnotes
    Support  Uren
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 3259. doi:
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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)

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Abstract

Purpose : 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.

Methods : 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.

Results : 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.

Conclusions : 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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