Fig. 4
Phagocytosis of photoreceptor outer segments by iPSC-RPE cells in vitro. Immunofluorescence labeling of iPSC-RPE cells for integrin αvβ5 (a) and MERTK (b). These receptors are responsible for the binding and internalization of POSs, respectively, and are present on the apical surface. c Image merge of a and b. The ability of iPSC-RPE cells to phagocytize POSs was tested in vitro by challenging the cells with POSs isolated from porcine retinas. d Micrograph of iPSC-RPE cells that have not been exposed to POSs and labeled with RHO antibody. Nuclei counterstained with DAPI. e Representative image of bound ROSs, labeled with an antibody against RHO and a green secondary antibody, prior to cell permeabilization. f Representative image of all (internalized and bound) ROSs labeled with the same RHO antibody, but with a red secondary antibody, following cell permeabilization. g When merged, the surface-bound ROSs appear yellow while internalized ROSs appear red. A few internalized ROSs are indicated by white arrowheads. h Quantification of bound and ingested ROSs was performed using the ImageJ software to count RHO-positive particles with diameters greater than 0.5 μm. i Total ROSs per field of view were quantified after the 2-h pulse, and after a 2-h and 5-h chase. Phagosome counts were obtained from six to eight individual fields of view, with each field containing ≥ 100 cells. h, i Data represent mean ± SD. Scale bars: a, d, 20 μm; e–g, 5 μm. ROS rod outer segment