title: Using flow cytometry to compare the dynamics of photoreceptor outer segment phagocytosis in iPS-derived RPE cells.
abstract: OBJECTIVE
  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 α(v)β₅ 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.
authors.affiliation: Department of Cell Biology, The Scripps Research Institute, La Jolla, California, USA.
authors.forename: Peter D
authors.initials: PD
authors.lastname: Westenskow
authors.forename: Stacey K
authors.initials: SK
authors.lastname: Moreno
authors.forename: Tim U
authors.initials: TU
authors.lastname: Krohne
authors.forename: Toshihide
authors.initials: T
authors.lastname: Kurihara
authors.forename: Saiyong
authors.initials: S
authors.lastname: Zhu
authors.forename: Zhen-Ning
authors.initials: ZN
authors.lastname: Zhang
authors.forename: Tongbiao
authors.initials: T
authors.lastname: Zhao
authors.forename: Yang
authors.initials: Y
authors.lastname: Xu
authors.forename: Sheng
authors.initials: S
authors.lastname: Ding
authors.forename: Martin
authors.initials: M
authors.lastname: Friedlander
mesh_terms: D000818:Animals; D005136:Eye Proteins / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D010587:Phagocytosis / Q000502:physiology*; D011971:Receptors, Immunologic / Q000378:metabolism; D055214:Retinal Photoreceptor Cell Outer Segment / Q000502:physiology*; D013552:Swine
chemical_list: D005136:Eye Proteins; D011971:Receptors, Immunologic; C028495:phagocytosis receptor