July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Near infra-red auto-fluorescence imaging of melanin in retinal pigment epithelium derived from induced pluripotent stem cells of albinism patients
Author Affiliations & Notes
  • Tyler Pfister
    NEI, CHEVY CHASE, Maryland, United States
  • Aman George
    NEI, CHEVY CHASE, Maryland, United States
  • Ruchi Sharma
    NEI, CHEVY CHASE, Maryland, United States
  • Rafael Villasmil
    NEI, CHEVY CHASE, Maryland, United States
  • Margery Smelkinson
    NIAID, Bethesda, Maryland, United States
  • Monika Barbara Dolinska
    NEI, CHEVY CHASE, Maryland, United States
  • Kapil Bharti
    NEI, CHEVY CHASE, Maryland, United States
  • Brian P Brooks
    NEI, CHEVY CHASE, Maryland, United States
  • Footnotes
    Commercial Relationships   Tyler Pfister, None; Aman George, None; Ruchi Sharma, None; Rafael Villasmil, None; Margery Smelkinson, None; Monika Dolinska, None; Kapil Bharti, None; Brian Brooks, None
  • Footnotes
    Support  NIH intramural
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 4663. doi:https://doi.org/
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    • Get Citation

      Tyler Pfister, Aman George, Ruchi Sharma, Rafael Villasmil, Margery Smelkinson, Monika Barbara Dolinska, Kapil Bharti, Brian P Brooks; Near infra-red auto-fluorescence imaging of melanin in retinal pigment epithelium derived from induced pluripotent stem cells of albinism patients. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4663. doi: https://doi.org/.

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

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Abstract

Purpose : Functional retinal pigment epithelium (RPE) cultures can be derived from induced pluripotent stem cells (iPSCs) to investigate the molecular mechanisms underlying pigmentation defects in vitro in models of human albinism. However, no well-defined method exists for quantification of pigment content in live cells. We aimed to develop robust, sensitive techniques to detect subtle changes in pigmentation by exploiting the near infrared (NIR) autofluorescence of melanin in RPE cells. We used flow cytometry and confocal microscopy for their potential to detect small differences in fluorescence intensities and for their scalability for high-throughput applications.

Methods : iPSC-RPE cells from unaffected individuals were used as a positive control, albinism patient-derived RPE with no pigmentation were used as negative control and RPE with varying degrees of pigmentation defects were the experimental group. Trypsinized cells were analyzed using Cytoflex® flow-cytometer (Beckman Coulter) equipped with avalanche photodiode detectors using an 808 nm excitation laser and 840/20 and 885/40 emission filters. RPE cultured in chamber slides for two weeks were used for live cell imaging using a confocal microscope equipped with a 730nm excitation laser and light-sensitive Hybrid detectors.

Results : We detected NIR autofluorescence from melanin in RPE with Cytoflex® flow-cytometer. High autofluorescence was observed in control pigmented RPE cells whereas, intermediate to no fluorescence was observed in differentially pigmented albinism RPE cells. Similarly, by live imaging of cells using confocal microscopy, we detected strong autofluorescence in control pigmented RPE, while albinism RPE showed variable to no auto fluorescence. The data was further confirmed by a conventional spectrophotometry-based assay upon cell lysis with NaOH followed by measurement of optical density at 475 nm.

Conclusions : Our data suggest that flow cytometry and live-cell imaging can provide precise measurements of melanin autofluorescence intensity. Furthermore, these techniques do not require fixation or antibody staining and can be performed on live cells either in suspension or in adherent culture. Their scalability makes both techniques attractive as readout assays for high throughput small molecule screens, especially in developing novel treatments for albinism.

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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