Comprehensive Analysis of Photoreceptor Outer Segments: Flow Cytometry Characterization and Stress-Driven Impact on Retinal Pigment Epithelium Phagocytosis

Phagocytosis is one of the key functions of retinal pigment epithelium (RPE) cells, which maintain photoreceptor health by removing photoreceptor outer segments (POSs) that are regularly shed. A deficiency in RPE function to phagocytose POSs may lead to vision loss in inherited retinal diseases and...

Full description

Saved in:
Bibliographic Details
Published inInternational journal of molecular sciences Vol. 24; no. 16; p. 12889
Main Authors Liang, Haoqian, Wu, Qiang, Guo, Xinzheng Victor, Chan, Linda, Mao, Tin, Stella, Cinzia, Guilbaud, Axel, Camperi, Julien
Format Journal Article
LanguageEnglish
Published Basel MDPI AG 01.08.2023
MDPI
Subjects
Biotechnology industry
Care and treatment
Dyes
Epithelium
Flow cytometry
fluorescence scanning
Macular degeneration
Microscopy
phagocytosis assay
photoreceptor outer segments
Photoreceptors
Retina
retinal pigment epithelium cells
Rhodopsin
Stem cells
Transplantation
United States
California
Online AccessGet full text

Cover

More Information
Summary:Phagocytosis is one of the key functions of retinal pigment epithelium (RPE) cells, which maintain photoreceptor health by removing photoreceptor outer segments (POSs) that are regularly shed. A deficiency in RPE function to phagocytose POSs may lead to vision loss in inherited retinal diseases and eventually to age-related macular degeneration (AMD) with geographic atrophy. Significant progress has been made in the field of cell replacement therapy for AMD using stem-cell-derived RPE. To test their function, RPE cells are incubated with purified bovine POSs for the demonstration of efficient binding, internalization, and digestion of POSs. Here, we present an image-based method to measure phagocytosis activity by using POSs labeled with a pH-sensitive fluorescent dye, which has low fluorescence at neutral pH outside of the cell and high fluorescence at low pH inside the phagosome. Further, we introduce a unique flow-cytometry-based method for the characterization of POSs by measuring specific markers for POSs such as rhodopsin and opsin. Using this method, we demonstrated a comparable quality of several bovine POS isolation batches and a reliable assessment of POS quality on RPE phagocytosis assay performance when subjected to different stress conditions. This work provides new tools to characterize POSs and insight into RPE phagocytosis assay development for the functional evaluation of RPE cells in the field of cell replacement therapy.
Bibliography:ObjectType-Article-1
SourceType-Scholarly Journals-1
ObjectType-Feature-2
content type line 23
These authors contributed equally to this work.
ISSN:1422-0067
1661-6596
1422-0067
DOI:10.3390/ijms241612889