June 2020
Volume 61, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2020
Characterization of the ageing of human primary RPE cells using single-cell RNA sequencing
Author Affiliations & Notes
  • Imre Lengyel
    Wellcome-Wolfson Institute for Experimental Medicine, Queen's University Belfast, Belfast, United Kingdom
  • Oisín Cappa
    Wellcome-Wolfson Institute for Experimental Medicine, Queen's University Belfast, Belfast, United Kingdom
  • David A Simpson
    Wellcome-Wolfson Institute for Experimental Medicine, Queen's University Belfast, Belfast, United Kingdom
  • Eszter Emri
    Wellcome-Wolfson Institute for Experimental Medicine, Queen's University Belfast, Belfast, United Kingdom
  • Footnotes
    Commercial Relationships   Imre Lengyel, Hoffman La Roche (F), OPTOS (C), Oxurion (F); Oisín Cappa, None; David Simpson, None; Eszter Emri, Hoffman La Roche (F)
  • Footnotes
    Support  EU Horizon2020
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 2269. doi:
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    • Get Citation

      Imre Lengyel, Oisín Cappa, David A Simpson, Eszter Emri; Characterization of the ageing of human primary RPE cells using single-cell RNA sequencing. Invest. Ophthalmol. Vis. Sci. 2020;61(7):2269.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Retinal pigment epithelial cells (RPE) in culture are used to model how cells behave in the human eye. When they are cultured for an extended period of time they can recapitulate aspects of the RPE in vivo, including accumulation of extracellular deposits, the hallmarks of age-related macular degeneration. Here we used single-cell sequencing (scRNA-Seq) to understand the molecular background behind cell heterogeneity and aging in long term cell cultures.

Methods : Human primary RPE (hpRPE) cells were cultured for 2, 9, and 19 weeks. Differentiation was assessed by monitoring pigmentation and transepithelial resistance. At the indicated times cells were dissociated to form single cell suspension and ~2000 cells per well were captured and processed in the Chromium System (10x Genomics, v3 single-cell reagent kit). Barcoded libraries were pooled and sequenced on Novaseq platform (Illumina). Data were analysed with CellRanger version 3.0.0 and Seurat (version 3.1). Adjacent wells were fixed for further analysis for mineralisation (osteosense) and immunostaining (ZO-1, RPE65, PMEL17, COL1A1) and electron microscopy

Results : hpRPE cells developed heterogeneous pigmentation which gradually increased with age. Mineralization, one of the features of drusen, started to occur after 9 weeks in culture. TER values were 2W: 112.9±3.9; 9w: 195.2±16.6; 19W: 151.8 ±7.4 ohm*cm2. Analysis of scRNA-seq transcriptome data revealed two distinct populations of RPE cells, both expressing markers of differentiated RPE, but one characterised by high expression of COL1A1 and the other by high expression of RPE65 and TTR. The ratio of the two cell populations changed over time from 52.4% at 2W to 67.1% at 19W. Both cell population expressed PMEL-17, the precursor of pigmentation, suggesting that with time all cells probably would develop the typical RPE phenotype.

Conclusions : hpRPE cells in culture can develop into a fully differentiated pigmented epithelium with expression of molecules usually involved in mineralisation coincides with the appearance of mineral deposits, suggesting that long term culture provides a suitable model for modifying the course of end stage disease. The discovery of two populations of hpRPE cells in culture raises the possibility that this may be reflected in vivo. This insight into RPE cell heterogeneity may help understanding of the pathology of age-related macular degeneration.

This is a 2020 ARVO Annual Meeting abstract.

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