September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Drusen Composition and Analysis of patient derived hiPSC-RPE cultures
Author Affiliations & Notes
  • Chad Galloway
    Department of Ophthalmology and Biomedical Genetics, University of Rochester, Rochester, New York, United States
  • Sonal Dalvi
    Department of Ophthalmology and Biomedical Genetics, University of Rochester, Rochester, New York, United States
  • Leslie MacDonald
    Department of Ophthalmology and Biomedical Genetics, University of Rochester, Rochester, New York, United States
  • Ruchira Singh
    Department of Ophthalmology and Biomedical Genetics, University of Rochester, Rochester, New York, United States
    Center for Visual Science, University of Rochester, Rochester, New York, United States
  • Footnotes
    Commercial Relationships   Chad Galloway, None; Sonal Dalvi, None; Leslie MacDonald, None; Ruchira Singh, None
  • Footnotes
    Support  Brightfocus Foundation Macular Degeneration Research Grant, David Bryant Trust, Knight Templar Eye Foundation Research Starter Grant, Research to prevent Blindness (Career Development Award, Singh- PI), Research to Prevent Blindness (unrestricted grant, Feldon- PI) Retina Research Foundation Pilot Study Grant, University Research Award (University of Rochester).
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 6059. doi:
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    • Get Citation

      Chad Galloway, Sonal Dalvi, Leslie MacDonald, Ruchira Singh; Drusen Composition and Analysis of patient derived hiPSC-RPE cultures. Invest. Ophthalmol. Vis. Sci. 2016;57(12):6059.

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

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Abstract

Purpose : Retinal pigment epithelium cells serve as a physical protective barrier in the retina while regulating phagocytosis, the visual cycle and epithelial transport to and from the choroidal vasculature. It follows then that RPE dysfunction is associated with pathologies affecting vision, such as macular degeneration. The development of technologies to derive retinal pigment epithelium cells from patient inducible pluripotent stem cells (hiPSC) offers a platform to investigate mechanistic aspects of macular degenerative diseases in a controlled environment. However, to justify the use of hiPSC-RPE in the study of these diseases their ability to recapitulate in vivo pathologies must be confirmed. Thus, our purpose in this study was to determine if the accumulation of lipoproteinatous drusen infiltrates, a hallmark of maculopathies, could be recapitulated in patient-derived hiPSC-RPE cultures.

Methods : hiPSC-RPE derived from patients with various macular degenerative diseases and unaffected controls were grown in culture for 60-90 days. Mature monolayers of patient and control hiPSC-RPE in their native condition or after exposure to specific stressors (e.g. POS, human serum) were subsequently fixed, sectioned and characterized by 1) periodic acid schiff (PAS staining) and 2) immunohistochemistry for drusen resident proteins (e.g. ApoE, TIMP3, C3, C5B-9 complex). Furthermore, in a subset of experiments, drusen was dissected out of un-fixed hiPSC-RPE culture and characterized for protein expression.

Results : In patient-derived hiPSC-RPE cultures from several maculopathies, drusen-like particles were observed by PAS staining as infiltrates on the basal surface of RPE monolayers, above a primordial basement membrane, in hiPSC-derived RPE. The structures observed by PAS staining contained characteristic drusen resident proteins such as ApoE and TIMP3, visualized by immuno- fluorescence microscopy. Removal of RPE monolayers in culture revealed apparent aggregate structures similarly reactive to drusen proteins like, TIMP3 and ApoE.

Conclusions : hiPSC-RPE derived from patients with distinct maculopathies recapitulates a critical pathologic feature of in culture, the development of drusen. Further investigation will be necessary to examine the complete proteomic overlap between patient isolated and hiPSC-RPE isolated drusen.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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