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

Peter D. Westenskow, Stacey K. Moreno, Tim U. Krohne, Toshihide Kurihara, Saiyong Zhu, Zhen Ning Zhang, Tongbiao Zhao, Yang Xu, Sheng Ding, Martin Friedlander

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

Purpose. 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). Methods. 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 αvβ5 integrin, CD36, and MerTK receptors, which are required for phagocytosis, were compared. Results. 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. Conclusions. 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.

Original languageEnglish
Pages (from-to)6282-6290
Number of pages9
JournalInvestigative Ophthalmology and Visual Science
Volume53
Issue number10
DOIs
Publication statusPublished - 2012 Sep

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Retinal Pigment Epithelium
Phagocytosis
Flow Cytometry
Fluorescein-5-isothiocyanate
Transplants
Induced Pluripotent Stem Cells
Photoreceptor Cells
Integrins

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience
  • Medicine(all)

Cite this

Westenskow, P. D., Moreno, S. K., Krohne, T. U., Kurihara, T., Zhu, S., Zhang, Z. N., ... Friedlander, M. (2012). Using flow cytometry to compare the dynamics of photoreceptor outer segment phagocytosis in iPS-derived RPE cells. Investigative Ophthalmology and Visual Science, 53(10), 6282-6290. https://doi.org/10.1167/iovs.12-9721

Using flow cytometry to compare the dynamics of photoreceptor outer segment phagocytosis in iPS-derived RPE cells. / Westenskow, Peter D.; Moreno, Stacey K.; Krohne, Tim U.; Kurihara, Toshihide; Zhu, Saiyong; Zhang, Zhen Ning; Zhao, Tongbiao; Xu, Yang; Ding, Sheng; Friedlander, Martin.

In: Investigative Ophthalmology and Visual Science, Vol. 53, No. 10, 09.2012, p. 6282-6290.

Research output: Contribution to journalArticle

Westenskow, PD, Moreno, SK, Krohne, TU, Kurihara, T, Zhu, S, Zhang, ZN, Zhao, T, Xu, Y, Ding, S & Friedlander, M 2012, 'Using flow cytometry to compare the dynamics of photoreceptor outer segment phagocytosis in iPS-derived RPE cells', Investigative Ophthalmology and Visual Science, vol. 53, no. 10, pp. 6282-6290. https://doi.org/10.1167/iovs.12-9721
Westenskow, Peter D. ; Moreno, Stacey K. ; Krohne, Tim U. ; Kurihara, Toshihide ; Zhu, Saiyong ; Zhang, Zhen Ning ; Zhao, Tongbiao ; Xu, Yang ; Ding, Sheng ; Friedlander, Martin. / Using flow cytometry to compare the dynamics of photoreceptor outer segment phagocytosis in iPS-derived RPE cells. In: Investigative Ophthalmology and Visual Science. 2012 ; Vol. 53, No. 10. pp. 6282-6290.
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AU - Zhu, Saiyong

AU - Zhang, Zhen Ning

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AU - Xu, Yang

AU - Ding, Sheng

AU - Friedlander, Martin

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N2 - Purpose. 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). Methods. 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 αvβ5 integrin, CD36, and MerTK receptors, which are required for phagocytosis, were compared. Results. 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. Conclusions. 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.

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