June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
The human RPE-choroid in short term organ culture: evaluation of gene expression changes
Author Affiliations & Notes
  • Robert F Mullins
    Ophthalmology and Visual Sciences, University of Iowa , Iowa City, Iowa, United States
    Wynn Institute for Vision Research, Iowa City, Iowa, United States
  • S Scott Whitmore
    Ophthalmology and Visual Sciences, University of Iowa , Iowa City, Iowa, United States
    Wynn Institute for Vision Research, Iowa City, Iowa, United States
  • Kathleen R Chirco
    Ophthalmology and Visual Sciences, University of Iowa , Iowa City, Iowa, United States
    Wynn Institute for Vision Research, Iowa City, Iowa, United States
  • Grefachew Workalemahu
    Ophthalmology and Visual Sciences, University of Iowa , Iowa City, Iowa, United States
    Wynn Institute for Vision Research, Iowa City, Iowa, United States
  • Shemin Zeng
    Ophthalmology and Visual Sciences, University of Iowa , Iowa City, Iowa, United States
    Wynn Institute for Vision Research, Iowa City, Iowa, United States
  • Adam P DeLuca
    Ophthalmology and Visual Sciences, University of Iowa , Iowa City, Iowa, United States
    Wynn Institute for Vision Research, Iowa City, Iowa, United States
  • XiuYing Liu
    Ophthalmology and Visual Sciences, University of Iowa , Iowa City, Iowa, United States
    Wynn Institute for Vision Research, Iowa City, Iowa, United States
  • Jessica A Penticoff
    Ophthalmology and Visual Sciences, University of Iowa , Iowa City, Iowa, United States
    Wynn Institute for Vision Research, Iowa City, Iowa, United States
  • Luke A Wiley
    Ophthalmology and Visual Sciences, University of Iowa , Iowa City, Iowa, United States
    Wynn Institute for Vision Research, Iowa City, Iowa, United States
  • Edwin M Stone
    Ophthalmology and Visual Sciences, University of Iowa , Iowa City, Iowa, United States
    Wynn Institute for Vision Research, Iowa City, Iowa, United States
  • Budd Tucker
    Ophthalmology and Visual Sciences, University of Iowa , Iowa City, Iowa, United States
    Wynn Institute for Vision Research, Iowa City, Iowa, United States
  • Footnotes
    Commercial Relationships   Robert Mullins, None; S Scott Whitmore, None; Kathleen Chirco, None; Grefachew Workalemahu, None; Shemin Zeng, None; Adam DeLuca, None; XiuYing Liu, None; Jessica Penticoff, None; Luke Wiley, None; Edwin Stone, None; Budd Tucker, None
  • Footnotes
    Support  NEI EY-024605, EY-026087, Research to Prevent Blindness, the Elmer and Sylvia Sramek Charitable Foundation
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 619. doi:
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    • Get Citation

      Robert F Mullins, S Scott Whitmore, Kathleen R Chirco, Grefachew Workalemahu, Shemin Zeng, Adam P DeLuca, XiuYing Liu, Jessica A Penticoff, Luke A Wiley, Edwin M Stone, Budd Tucker; The human RPE-choroid in short term organ culture: evaluation of gene expression changes. Invest. Ophthalmol. Vis. Sci. 2017;58(8):619.

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

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Abstract

Purpose : Robust in vitro systems, employed to study the effects of disease-associated environmental insults on cellular physiology, offer excellent opportunities to understand the pathology of diseases such as age-related macular degeneration. Previous studies by us and others have shown ultrastructural preservation of RPE-choroid organ cultures after several days. In the current study we compared gene expression in freshly isolated and cultured tissues and cells.

Methods : Human donor eyes were obtained and utilized for: (a) RPE isolation, followed by either flash freezing (n=3 samples, donor 1) or 7 days incubation on tissue culture plastic (n=3 samples, donor 1); or (b) RPE-choroid collection, followed by flash freezing (n=3 samples, donor 2) or 7 day organ culture (n=3 samples, donor 2). Total RNA was then isolated and transcript levels were determined using RNA-Seq (75 nucleotide paired-end reads of poly-A selected RNA). Sequencing reads were mapped to the human genome build hg19 using Tophat2 and quantified using Rsubread. Differential expression analysis was performed using edgeR.

Results : RPE cells in organ cultures showed more similarity to the RPE in vivo than did RPE cells that were plated on tissue culture plastic. When expression of the RPE-specific genes RPE65, BEST1, and RLBP1 was evaluated, levels in isolated RPE cells on tissue culture plastic were reduced to 2%, 3% and <1% of uncultured cell levels, whereas in organ cultures these levels were 65%, 83% and 25% of flash frozen samples, respectively. Cultured RPE-choroid also continued to express vascular endothelial cell-specific genes VWF, CDH5, CD34, and ICAM2 after 7 days of culture at similar levels to fresh tissue.

Conclusions : Human RPE-choroid organ cultures have relatively high fidelity of gene expression compared to primary cultures and retain viable RPE and endothelial cells at least up to 7 days in culture.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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