In culturing
KCNJ13-KO hiPSC-RPE cells at 2 and 4 weeks after seeding, we found that there were more protruded cells from the bottom of the culture dish compared with WT hiPSC-RPE cells. We counted the number of protruded cells per area and found that there was a significant increase in protruded cells in
KCNJ13-KO hiPSC-RPE cells (at 2 weeks, WT: 0.76 ± 0.12, KO: 13.56 ± 1.30,
P < 0.001, n = 5; at 4 weeks, WT: 0.32 ± 0.11, KO: 10.24 ± 2.36,
P < 0.001, n = 5) (
Figs. 3A,
3B). We observed cultured WT and
KCNJ13-KO hiPSC-RPE cells by confocal laser microscopy to determine the structure of the cells. We used rhodamine–phalloidin staining to visualize actin filaments and immunostaining with anti-RPE65 antibodies to identify the cytoplasm of RPE cells. The WT hiPSC-RPE cells formed a single epithelial cell layer (
Figs. 3C–
3G). By contrast, in
KCNJ13-KO hiPSC-RPE cells, a subpopulation of cells formed a round elevated layer from a surrounding RPE monolayer (
Figs. 3H–
3L). Because the protruded cell group was immunoreactive for RPE65 (
Figs. 3J,
3L), we concluded that they were differentiated RPE cells. To further see the structure of protruded RPE cells, we observed cultured hiPSC-RPE cells of WT and
KCNJ13-KO by transmission electron microscopy. As previously reported,
22 the WT hiPSC-RPE cells had elongated apical microvilli, apically distributed melanosomes, and basal membrane infoldings (
Supplementary Fig. S4A and not shown). In contrast, although similar microvilli, melanosomes, and basal structures were observed in the single-layered
KCNJ13-KO hiPSC-RPE cells (
Supplementary Fig. S4B), there were some portions of double-layered cells and cell debris clumping laterally and beneath the RPE cells (
Supplementary Fig. S4C).