Using flow cytometry to compare the dynamics of photoreceptor outer segment phagocytosis in iPS-derived RPE cells

Retinal pigment epithelium (RPE) autologous grafts can be readily derived from induced pluripotent stem (iPS) cells. It is critical to stringently characterize iPS-RPE using standardized and quantifiable methods to be confident that they are safe and adequate replacements for diseased RPE before uti...

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Bibliographic Details
Published in:Investigative ophthalmology & visual science 2012-09, Vol.53 (10), p.6282-6290
Main Authors: Westenskow, Peter D, Moreno, Stacey K, Krohne, Tim U, Kurihara, Toshihide, Zhu, Saiyong, Zhang, Zhen-Ning, Zhao, Tongbiao, Xu, Yang, Ding, Sheng, Friedlander, Martin
Format: Article
Language:English
Subjects:
Animals
Eye Proteins - metabolism
Flow Cytometry - methods
Humans
Induced Pluripotent Stem Cells - cytology
Phagocytosis - physiology
Receptors, Immunologic - metabolism
Retinal Photoreceptor Cell Outer Segment - physiology
Swine
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Summary:Retinal pigment epithelium (RPE) autologous grafts can be readily derived from induced pluripotent stem (iPS) cells. It is critical to stringently characterize iPS-RPE using standardized and quantifiable methods to be confident that they are safe and adequate replacements for diseased RPE before utilizing them in clinical settings. One important and required function is that the iPS-RPE phagocytose photoreceptor outer segments (POS). We developed a flow cytometry-based assay to monitor binding and internalization of FITC labeled POS by ARPE-19, human fetal RPE (hfRPE), and two types of iPS-RPE. Expression and density of α(v)β₅ integrin, CD36, and MerTK receptors, which are required for phagocytosis, were compared. Trypsinization of treated RPE cells results in the release of bound POS. The number of freed POS, the percentage of cells that internalized POS, the brightness of the FITC signal from the cells, and the surface density of the phagocytosis receptors on single RPE cells were measured using flow cytometry. These assays reveal that receptor density is dynamic during differentiation and this can affect the binding and internalization dynamics of the RPE cells. Highly differentiated iPS-RPE phagocytose POS more efficiently than hfRPE. Caution should be exercised to not use RPE grafts until demonstrating that they are fully functional. The density of the phagocytosis receptors is dynamic and may be used as a predictor for how well the iPS-RPE cells will function in vivo. The phagocytosis dynamics observed between iPS-RPE and primary RPE is very encouraging and adds to mounting evidence that iPS-RPE may be a viable replacement for dysfunctional or dying RPE in human patients.
ISSN:1552-5783
0146-0404
1552-5783
DOI:10.1167/iovs.12-9721